Antibodies, antibody fragments and analogs specific for chondroitin sulfate

Abstract

Antibodies and antibody variants that specifically bind to oncofetal chondroitin sulfate are provided. Also provided are conjugates, fusion proteins, and CAR-T cells that include the antibodies or antibody variants, as well as methods and uses of these agents for therapeutic and diagnostic purposes, particularly the treatment and diagnosis of cancer.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to antibodies and antibody variants that specifically target oncofetal chondroitin sulfate (ofCS), as well as means and methods for their preparation and use. [Background technology]

[0002] 2. Background of the Invention CSs belong to the glycosaminoglycan (GAG) family and are long unbranched disaccharide repeating units attached to a protein core organized as chondroitin sulfate proteoglycans (CSPGs). CSPGs can be located intracellularly, on the cell membrane, or in the extracellular matrix (ECM). CSPGs can contain one or multiple GAG ​​chains. GAGs are classified into six different classes based on the identity of the repeating disaccharide unit: chondroitin sulfate (CS, hereafter CSA, CSB, CSC), heparin, heparan sulfate (HS), keratin sulfate, and hyaluronic acid. Both CSA / C and CSB have the monosaccharide unit N-acetylgalactosamine (GalNAc) and a uronic acid residue, but this residue differs in chirality, being glucuronic acid (GlcA) in the case of CSA / C and iduronic acid (IdoA) in the case of CSB. GAG chains consisting mainly of CSB are sometimes called dermatan sulfate. CS disaccharide units can be modified by the addition of sulfate groups, such as sulfation at carbon 2 of GlcA or at carbons 4 (C4S) and 6 (C6S) of GalNAc. CSA has C4S, whereas CSC has C6SGalNAc modification. Further heterogeneity and specificity may arise from hybrid structures, for example, consisting of chains of CSA with islands of CSB dispersed within them. Here, we refer to CS as including CSA, CSB, CSC, and their hybrid structures. In particular, C4S and C6S type CSPGs (also called CSA / CSC) have been reported in various malignancies, whereas CSA / CSC-CSB hybrid chains, which reportedly provide optimal sequences for the binding of the growth factor pleiotrophin during embryonic development, have also been reported in various malignancies. Aberrant expression of CSPGs is directly related to most types of cancer. Chondroitin sulfate proteoglycan 4 (CSPG4), CD44 (CSPG8), and versican (CSPG2) are membrane CSPGs that promote malignant transformation. Specific CSPG expression patterns have also been observed in various cancers. For example, CSPG4 is highly expressed in many tumor cells. CD44 is one of the most commonly used biomarkers for breast cancer stem cells, and high levels of versican have been associated with various malignancies.CSPGs promote the growth, migration, and invasion of both primary tumors and metastases by binding to chemokines and by recruiting growth factor receptor complexes and extracellular matrix components to transduce pro-oncogenic signaling events. Thus, CSPGs have been widely exploited as targets for cancer therapeutic and diagnostic purposes. The main focus of therapeutic strategies has been on targeting the protein core of CSPGs, with little attention paid to the carbohydrate moiety of CSPGs. This is mainly due to the technical challenges of oligosaccharide characterization and the lack of antibodies capable of distinguishing between different CS structures. Some of the published and used CS antibodies bind only to the first GalNAc-GlcA stub (e.g., 2B6 or 3B3) attached to the protein core that appears after enzymatic chondroitinase treatment, and therefore are of no clinical relevance from a therapeutic targeting perspective (Davies et al., Osteoarthritis and Cartilage, 2008; Hardingham et al., Carbohydrate Research, 1994). Other CS antibodies, mainly low-affinity IgM (e.g., CS-56 or PG-4), recognize simple structures and have not been demonstrated to specifically target tumors in vivo (Detamore et al., Matrix Biology, 2005; Garcia-Piqueras et al., Anat. Rec. (Hoboken), 2019). Therefore, due to the lack of highly specific and high-affinity antibody reagents against tumor-associated glycomodifications, the focus has been on the various protein scaffolds themselves, as proteoglycans are often also overexpressed in cancers.

[0003] Focusing on the protein portion of CSPGs is problematic if malignant function relies on the nature of CS displayed by CSPGs. Malignant CS may be redundantly displayed on multiple different protein cores, limiting the potential of targeting a single protein. Furthermore, therapeutic resistance may result because CS, a pleiotropic and functional determinant, may shift to protein cores that are not therapeutic targets. A growing number of studies have shown that the function of CSPGs is exerted through the interaction of specific CS chain motifs with the core protein scaffold. Two examples are versican and CSPG4, which serve as ligands for P-selectin and L-selectin expressed on endothelial cells. These interactions are mediated by CS and may contribute to the tumor metastatic process. Another example is the role of CS in the activation of metalloprotease 2 (MMP-2), an ECM-degrading enzyme. MMP-2 activation has been associated with enhanced cancer invasiveness and metastasis, and 4-O-sulfated CS on CSPG4 in melanoma was found to enhance MMP-2 activation. Only a few CS motifs have been identified so far, which is mainly due to the limitations of structural analysis of specific GAG chains, which usually cannot provide information on the exact sequence but only on the overall disaccharide composition.

[0004] VAR2CSA-ofCS-binding malaria protein: Infection with the parasite Plasmodium falciparum during pregnancy can cause placental malaria. The parasite infects red blood cells, and these infected cells are filtered and removed in the spleen. To avoid removal, the parasite expresses proteins on the surface of infected red blood cells that effectively anchor these cells to the blood vessel wall. Parasites accumulate in the placenta because they can bind to CSPGs expressed in placental syncytiotrophoblasts. The parasite protein that mediates the binding, VAR2CSA, has evolved to promote high affinity binding to CSPGs, including CS, in the placenta. Applicants discovered VAR2CSA in 2003 and subsequently mapped and characterized the CS-binding domain of VAR2CSA. A recombinant subfragment of VAR2CSA (rVAR2) binds CS with affinity in the low nanomolar range (1-10 nM). The binding is highly specific, as rVAR2 does not bind to carbohydrates with sulfation patterns different from those found in placental CS. This specific interaction to different placental CS is the result of natural selection of VAR2CSA-expressing parasites that bind to placental tissue, as this is the only placental tissue where this particular CS is found in the vasculature. Thus, parasites expressing VAR2CSA are sequestered exclusively in the placenta. The placenta is a rapidly growing organ, whose cells exhibit high mitotic rates, the ability to invade uterine tissue, and the ability to evade the immune system. CSPGs, including CS, are abundantly expressed by trophoblasts, attracting chemokines and nutrients to ensure a high division rate of placental cells. The expression of CSPGs in placental cells is thought to reflect the unique morphology of CS chains, a requirement for these cells to migrate and divide rapidly, a feature shared with cancer cells. We therefore speculated that cancer cells turn on the expression of unique oncofetal CS (ofCS) to promote rapid growth and metastatic spread.

[0005] Applicants tested the binding of rVAR2 to hundreds of cancer cell lines and found that rVAR2 reacted with over 95% of patient-derived human cancer cell lines, whereas non-cancerous cells did not bind rVAR2. Applicants tested a series of human tumors compared to matched adjacent normal tissues from the same patients for rVAR2 binding. All tumors showed strong staining for rVAR2, whereas matched normal tissues showed only weak staining. Binding of rVAR2 to primary human tumors could be completely inhibited by enzymatic removal of CS from the tissues. In a larger cohort, rVAR2 tissue binding strongly correlated with progression in patients with malignant melanoma, poor recurrence-free survival in patients with non-small cell lung cancer, and poor outcome in patients with bladder cancer. Mass spectrometry analysis of rVAR2 pulldowns from cancer cells and tissues demonstrates that a variety of proteins, particularly those involved in proliferation and cell migration, are modified by ofCS glycosylation.

[0006] When fluorescently labeled rVAR2 is injected intravenously (IV) into mice bearing various tumors (xenografts, allografts, PDX), rVAR2 rapidly localizes to tumor tissue. Similarly, IV administration of rVAR2-drug conjugates is not associated with side effects and has been shown to shrink tumors in various experimental models, including prostate cancer, orthotopic bladder cancer, lymphoma, metastatic breast cancer, melanoma, and orthotopic PDX models of glioblastoma. Testing of blood samples from cancer patients showed that rVAR2 immobilized on magnetic beads can capture circulating tumor cells (CTCs) even at very early stages from blood samples of all cancer patients tested (pancreatic cancer, prostate cancer, lung cancer, glioma, melanoma, breast cancer, liver cancer). Applicants have developed a technology to capture circulating tumor cells (CTCs) in blood samples using magnetic beads coated with rVAR2. These data demonstrated ubiquitous cancer cell expression of ofCS and the clinical feasibility of targeting ofCS (Salanti and Clausen et al., Cancer Cell, 2015; Agerbaek et al., Nat. Commun., 2018).

[0007] Importantly, these clinical data indicate widespread cancer cell expression of ofCS. Published data show important information about ofCS structure. In summary, these data show that ofCS consists of a complex hybrid structure of both 4 (CSA) and 6 (CSC) sulfated CS and IdoA molecules (i.e., dermatan sulfate DS or CSB). Furthermore, data demonstrate that ofCS chains associated with placental development and cancer are aberrantly overexpressed and particularly long. This is consistent with footprint analysis of the rVAR2-ofCS binding interface, indicating that rVAR2 binds long oligosaccharide chains, which likely determine oncofetal nutrition (Spliid et al., J. Biol. Chem., 2021).

[0008] In summary, ofCS is highly expressed both on the surface of cancer cells and in the ECM of tumor tissues, and is absent in normal tissues, making it an attractive cancer target. Clearly, rVAR2 can be used to target ofCS to cancer and cancer cells, and this protein could potentially form the basis of new drugs and diagnostics. However, using recombinant malaria VAR2 protein for therapeutic purposes has proven difficult. When injected intravenously, the plasma half-life of the recombinant protein is less than 5 minutes due to liver clearance. Thus, treating tumors may require large amounts of protein and repeated injections. Another consideration is that rVAR2 is a foreign malaria protein, and at some point, an antibody response will be induced that may neutralize the treatment. Finally, recombinant malaria proteins are large and complex proteins with hundreds of cysteines and many interdomain disulfide bonds, making production and stability a challenge. Antibodies with the same specificity as rVAR2 would avoid the risks associated with the production, immunogenicity, and serum half-life of rVAR2. Anti-ofCS monoclonal antibodies (mAbs) may be effective by themselves, even without toxin conjugation, because they persist in plasma for extended periods and may neutralize CTCs during migration to the premetastatic niche.

[0009] Objective of the invention It is an object of embodiments of the present invention to provide antibodies and antibody variants that specifically bind to ofCS and to provide means, methods and uses for the treatment and diagnosis of cancer. Summary of the Invention

[0010] Conventional antibodies against CS are of the IgM type, generally have low affinity, and are unable to distinguish ofCS overexpressed in cancer cells from other glycosaminoglycans such as heparan sulfate, hyaluronic acid, or simple CSA structures. However, the inventors have found that all of the antibodies and antibody variants provided herein have high specificity for ofCS and thereby the ability to distinguish ofCS from other GAGs, and as a result, their binding properties are comparable to those of VAR2CSA.

[0011] Thus, in a first aspect, the present invention relates to an antibody or antibody variant that specifically binds to chondroitin sulfate (CS) glycosaminoglycan chains, which antibody or antibody variant exhibits a higher binding affinity to oncofetal CS (ofCS) than to non-oncofetal glycosaminoglycans such as heparan sulfate and hyaluronic acid.

[0012] In a second aspect, the present invention relates to a conjugate or fusion protein comprising at least a first and a second moiety, wherein the first moiety is an antibody or antibody variant according to the first aspect of the invention or any embodiment thereof disclosed herein and the second moiety is a molecule or polypeptide, the first moiety being conjugated or genetically fused to the second moiety and the second moiety providing or improving a therapeutic and / or diagnostic function of the conjugate or fusion protein.

[0013] In a third aspect, the present invention is i) a chimeric antigen receptor (CAR) transmembrane domain and endodomain.

[0014] In a fourth aspect, the present invention relates to a CAR-T cell comprising a polypeptide of the third aspect of the invention or any embodiment thereof disclosed herein.

[0015] In a fifth aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, a conjugate or fusion protein of the second aspect of the invention or any embodiment thereof disclosed herein, or a polypeptide of the third aspect of the invention or any embodiment thereof disclosed herein.

[0016] In a sixth aspect, the present invention relates to a vector, such as an expression vector or a cloning vector, comprising the isolated nucleic acid molecule of the fifth aspect of the invention or any embodiment thereof disclosed herein.

[0017] In a seventh aspect, the present invention relates to a host cell comprising or transformed with a vector of the sixth aspect of the invention or any embodiment thereof disclosed herein.

[0018] In an eighth aspect, the present invention relates to a method for producing an antibody or antibody variant according to the first aspect of the invention or any embodiment thereof disclosed herein, a conjugate or fusion protein according to the second aspect of the invention or any embodiment thereof disclosed herein, or a polypeptide according to the third aspect of the invention or any embodiment thereof disclosed herein, comprising the steps of transfecting or transforming a host cell with a vector according to the sixth aspect of the invention or any embodiment thereof disclosed herein, expressing a nucleotide sequence according to the fifth aspect of the invention or any embodiment thereof disclosed herein, and isolating the antibody or antibody variant, conjugate or fusion protein, or polypeptide.

[0019] In a ninth aspect, the present invention relates to a pharmaceutical composition comprising an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, a conjugate or fusion protein of the second aspect of the invention or any embodiment thereof disclosed herein, or a CAR-T cell of the fourth aspect of the invention or any embodiment thereof disclosed herein, and a pharma- ceutically acceptable carrier, vehicle or diluent.

[0020] In a tenth aspect, the present invention relates to the use of the antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, or the conjugate or fusion protein of the second aspect of the invention or any embodiment thereof disclosed herein, for in vitro detection and / or isolation of cancer cells, such as cancer cells derived from a subject, such as circulating tumor cells.

[0021] In an eleventh aspect, the present invention relates to an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, a conjugate or fusion protein of the second aspect of the invention or any embodiment thereof disclosed herein, a CAR-T cell of the fourth aspect of the invention or any embodiment thereof disclosed herein, or a pharmaceutical composition of the ninth aspect of the invention or any embodiment thereof disclosed herein, for use as a medicament.

[0022] In a twelfth aspect, the present invention relates to the antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, the conjugate or fusion protein of the second aspect of the invention or any embodiment thereof disclosed herein, the CAR-T cell of the fourth aspect of the invention or any embodiment thereof disclosed herein, or the pharmaceutical composition of the ninth aspect of the invention or any embodiment thereof disclosed herein, for use in diagnosing cancer in a subject.

[0023] In a thirteenth aspect, the present invention relates to the antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, the conjugate or fusion protein of the second aspect of the invention or any embodiment thereof disclosed herein, the CAR-T cell of the fourth aspect of the invention or any embodiment thereof disclosed herein, or the pharmaceutical composition of the ninth aspect of the invention or any embodiment thereof disclosed herein for use in the treatment of cancer in a subject in need thereof, or for the treatment of a condition involving expression (such as inappropriate expression) of ofCS, such as a condition selected from the group consisting of arthritis, arthropathy, multiple sclerosis, healing after nerve injury, cartilage repair, wound healing, and psoriasis, or for delivering a therapeutic to the placenta in a subject in need thereof.

[0024] In a fourteenth aspect, the invention relates to a method of treating cancer, or a condition involving expression (e.g. inappropriate expression) of ofCS, such as a condition selected from the group consisting of arthritis, arthropathy, multiple sclerosis, healing after nerve injury, cartilage repair, wound healing, and psoriasis, or a method of delivering a therapeutic to the placenta, comprising administering to a subject a therapeutically effective amount of an antibody or antibody variant of the first aspect of the invention, or any embodiment thereof disclosed herein, a conjugate or fusion protein of the second aspect of the invention, or any embodiment thereof disclosed herein, a CAR-T cell of the fourth aspect of the invention, or a pharmaceutical composition of any embodiment thereof disclosed herein.

[0025] In a fifteenth aspect, the present invention provides a method for detecting ofCS in a sample, the method comprising: 1) contacting said sample with an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, to allow said antibody or antibody variant to specifically bind to ofCS, and subsequently contacting said antibody or antibody variant and ofCS bound thereby with an agent capable of binding to ofCS, to allow said agent to bind to ofCS, or 2) contacting said sample with an agent capable of binding to ofCS, allowing said agent to bind to ofCS, and subsequently contacting said agent and the ofCS bound thereby with an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, allowing said antibody to bind to ofCS; and quantitatively or qualitatively detecting the presence of a complex comprising ofCS, said antibody or antibody variant, and said agent. wherein said agent and said antibody or antibody variant are non-competitive for binding to ofCS.

[0026] In a sixteenth aspect, the present invention relates to a detection kit comprising an antibody of the first aspect of the invention or any embodiment thereof disclosed herein, an agent as defined in the fourteenth aspect of the invention or any embodiment thereof disclosed herein, and a reaction vessel, and optionally also a labelling agent which binds to the antibody or the agent.

[0027] In a seventeenth aspect, the present invention relates to a method of providing a nucleic acid encoding an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, comprising screening a library comprising antigen-binding fragments of antibodies, the library comprising a display agent selected from cells, viruses and phages comprising nucleic acid fragments encoding the antibodies, allowing the display agent to bind to a capture agent consisting essentially of purified ofCS or a protein decorated with ofCS, subsequently isolating from the display agent the nucleic acid fragments encoding the antibodies that bind to the capture agent and optionally sequencing the isolated nucleic acid fragments.

[0028] Finally, in an eighteenth aspect, the invention relates to a method of producing an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, comprising expressing in a host cell in culture an expression vector comprising a nucleic acid fragment obtained according to the method of the sixteenth aspect of the invention or any embodiment thereof disclosed herein, and subsequently isolating the expression product from the culture. [Brief description of the drawings]

[0029] Text description of the illustration image024.gif.

[0030] [Figure 1] Binding of selected ofCS IgG antibodies to ofCS with and without chondroitinase treatment (no B3 binding was detected after chondroitinase treatment), measured by ELISA. [Diagram 2] Binding of specific ofCS scFv antibodies to ofCS with or without chondroitinase treatment measured by ELISA. [Diagram 3] Binding of ofCS antibody B3 and a commercially available anti-CS antibody to ofCS, ofCSPGs on the decorin backbone, and commercially available purified cartilage decorin was measured by ELISA. [Figure 4] Binding of select ofCSIgG antibodies to lymphoma cancer cells (Karpas) in the presence and absence of soluble CSA (binding of F2, F11, B3, and VAR2CSA is not detected in the presence of soluble CSA) as measured by flow cytometry normalized to background binding of the secondary antibody. [Diagram 5] Bladder cancer, placenta, and noncancerous liver tissue sections were stained with ofCSscFv antibody variant C9 and the presence or absence of chondroitinase processing (C-ase) was measured by immunohistochemistry (IHC). [Figure 6] Intravital imaging system (IVIS) CT images of two mice implanted with tumors in the right flank. The images show the distribution of NIR-labeled ofCSIgGC9 at different time intervals after tail vein injection. Clearly, the anti-ofCS antibodies are localized to the tumor implanted in the right flank of the mice. The mouse on the right was not injected with the antibody but was included to measure autofluorescence. [Figure 7]After preincubation with 50 μg C9 IgG, control antibody, or PBS, immunocompetent mice were injected with 200,000 luciferase-positive 4T1 tumor cells. After luciferin injection, lung tumor growth was monitored using IVIS scanning, and tumor incidence was plotted as a survival curve adjusted for the percentage risk of developing a tumor according to the treatment regimen. [Figure 8] Mice were injected with 4T1 tumor cells and treated peritumorally (PT) with VAR2-anti-CD3 or ofCSscFv(C9)-anti-CD3 bispecific molecules or PBS. The figure shows tumor growth over time. [Figure 9] Different amounts of CSPG rEndocan were spiked into the buffer and the ability of selected ofCSscFvs to capture CSPGs was measured by ELISA in which captured endocan was detected with an anti-endocan antibody. [Figure 10] Line graph showing tumor volume of CT26 colon carcinoma in control (left) and antibody C9 (right) treated mice. [Figure 11] Line graph showing tumor volume of Karpas lymphoma tumors in mice treated with control (left) and antibody C9 (right). [Figure 12] Tumor and normal matched tissues stained with DAPI (panels A and B) and antibody C9 (panels C and D). [Figure 13] Graph showing fluorescence from tumor cells treated with spyCAR+ T cells and C9 scFv, or non-transduced T cells and C9 scFv. [Figure 14-1] Schematic diagram of various construct formats, each containing the C9 CDR (sequence numbers 122-127), and corresponding graphs showing KD values ​​and binding kinetic curves. [Figure 14-2] Schematic diagram of various construct formats, each containing the C9 CDR (sequence numbers 122-127), and corresponding graphs showing KD values ​​and binding kinetic curves.

[0031] Detailed Disclosure of the Invention definition An "antibody", also called an "immunoglobulin", is a protein consisting of paired identical light and heavy chains in humans, each of which contains a variable domain and a light domain, where the variable domain is responsible for the specific binding of the antibody to the antigen. Generally, antibodies are classified into immunoglobulin groups IgG, IgM, IgA, IgE, and IgD, each of which plays a different role as an antigen recognition agent in the immune system. The specificity of the antibody's antigen binding is determined by the variable regions in the variable domain, especially the complementarity determining regions (CDRs) in the variable domain. In general, antibodies are molecules that in principle or in practice may be the product of natural expression in animals, i.e., they contain all the structural elements found in natural immunoglobulins.

[0032] An "antibody variant" is an antibody-derived protein that has the same binding specificity as an antibody, but is not a product of natural expression in a mammal. The term therefore refers to various fragments of antibodies and artificial antibody analogue formats. The term also refers to antibody formats that exist in nature but are not common in mammals, such as the single-chain antibodies known from llamas and camels, and IgY found in birds and reptiles, but in which the CDRs of a mammalian antibody or a combined generated antibody have been engineered into an antibody format to which they were not originally derived.

[0033] Oncofetal chondroitin sulfate (ofCS) is a form of chondroitin sulfate that is generally highly sulfated and is found in placental tissues and in many cancer cells. Its characteristics include high sulfation of GalNAc residues in the CS chain, especially with a majority of disaccharides bearing 4-O or 6-O sulfate groups on GalNAc, but is often (though not always) also sulfated with at least one unsulfated GalNAc and the presence of 6-O sulfated GalNAc and L-iduronic acid units. ofCS may also have fully 6-O sulfated GalNAc and no L-iduronic acid. The second unit of the ofCS disaccharide is either GlcA or IdoA (L-iduronic acid), and these structures, parallel to the ofCS chain, may exist as alternating hybrid structures or islands of CSA / C and CSB. Further complexity of the ofCS structure may consist of 2-O sulfation of GalNAc or IdoA sugars.

[0034] Specific embodiments of the present invention First aspect of the invention and embodiments thereof This aspect relates to an antibody or antibody variant that specifically binds to chondroitin sulfate (CS) glycosaminoglycan chains, wherein the antibody or antibody variant exhibits a higher binding affinity for oncofetal CS (ofCS) than for non-oncofetal glycosaminoglycans such as heparan sulfate and hyaluronic acid.

[0035] A preferred antibody of the first aspect comprises: - competes with VAR2CSA (SEQ ID NO: 135) or a CS-binding fragment thereof (such as the polypeptide having SEQ ID NO: 136) for binding to CS glycosaminoglycan chains; and / or 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, 121, and 128 for binding to -CS glycosaminoglycans. In other words, the antibody of the present invention is preferably an antibody having the antibody specificity exemplified herein and / or the same binding specificity for CS as VAR2CSA. As demonstrated in the examples, the exemplified antibodies and antibody variants do not all compete for binding to the same epitope on ofCS, which provides the advantage of targeting different epitopes on ofCS, which not only provides advantages in many immunoassay formats, but also opens the door to therapies that can utilize multiple targets on ofCS simultaneously.

[0036] As also shown in the Examples, the antigen binding site of the identified antibody is characterized by a characteristic positively charged groove, and therefore the binding site of the antibody or variant, in important embodiments, is V H / V L It is characterized by the presence of a positively charged groove parallel to the boundary.

[0037] The positively charged groove is V L and V H The domain may be comprised of positively charged, surface-exposed amino acid residues (i.e., Lys, Arg, or His) present in both variable loops, typically 3, 4, 5, or 6 positively charged amino acid residues. H / V L The distance between the α-carbon atoms of positively charged amino acids crossing the boundary is preferably at most 12 Å, for example in the range of 6-12 Å, preferably 6.7-11.4 Å. See the Examples for details.

[0038] As shown herein, all of the exemplified antibodies exhibit higher affinity for ofCS than the commercially available CS antibodies 2H6, CS56, BE-123, and PG-5. This is a characteristic of the antibodies and variants of the present invention, and this difference in affinity can be effectively used to distinguish ofCS-specific antibodies from known CS antibodies that are not used as broad cancer diagnostics or broad therapeutic agents. Furthermore, it is believed that some of the low affinity for ofCS exhibited by these commercially available antibodies is due to the fact that they are all IgM antibodies, and therefore, it is preferred that the antibodies of the present invention are different from IgM.

[0039] Another feature of the antibodies of the first aspect of the invention is their ability to bind directly to native ofCS, whereas at least some of 2H6, CS56, BE-123, and PG-5 require prior treatment of the ofCS with chondroitinase. Thus, preferred antibodies and variants of the first aspect bind to native ofCS and therefore do not require treatment with chondroitinase to bind with high affinity.

[0040] The antibody or antibody variant of the first aspect can in principle have any relevant format (i.e. IgG, IgE, IgD and IgA, and with the above reservation IgM format), but it is particularly preferred that the antibody or variant is an IgG antibody or is derived from an IgG antibody. IgG can be of any subclass, i.e. IgG1, IgG2, IgG3 and IgG4. In any case, the preferred antibody or variant is a human antibody or is derived from a human antibody, so that the antibody is preferably a human antibody, a humanized antibody or an antibody variant with pharma- ceutically acceptable low immunogenicity when administered to humans.

[0041] In line with the above considerations, a preferred antibody or variant of the first aspect is one that competes with a second antibody for binding to ofCS, the second antibody having a heavy chain variable region (V) having an amino acid sequence present in any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, 121, and 128. H 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, 121, and 128. L ).

[0042] In a particularly preferred embodiment, the antibody or antibody variant of the first aspect comprises H an amino acid sequence that is present in or is identical to an amino acid stretch C-terminally bounded by the sequence GGGGGSGGGGSGGGSGGGGS (SEQ ID NO: 137) of any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, and 128; or having an amino acid sequence identical to or present in an amino acid stretch C-terminally bound to the sequence GGGGSGGGGSGGGGSGGG of SEQ ID NO: 121 (SEQ ID NO: 138); and / or Here, V L The area is - an amino acid sequence that is present or identical to the amino acid sequence linked to the N-terminus of any of the sequences GGGGSGGGGSGGGSGGGGGS (SEQ ID NO: 137) of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, and 128, or - having an amino acid sequence that is present or identical to the amino acid sequence linked to the N-terminus of the sequence GGGGSGGGGSGGGGSGGG of SEQ ID NO: 121 (SEQ ID NO: 138).

[0043] In some preferred embodiments closely related to the Examples, the antibody or antibody variant comprises a light chain complementarity determining region (LCDR)1 having the sequence of SEQ ID NO: 2, 9, 16, 23, 30, 37, 44, 52, 59, 66, 73, 80, 87, 94, 101, 108, 115, 122, or 129, or a sequence having at least 70% sequence identity to any of these sequences, and an LCDR2 having the sequence of SEQ ID NO: 3, 10, 17, 24, 31, 38, 45, 53, 60, 67, 74, 81, 88, 95, 102, 109, 116, 123, or 130, or a sequence having at least 70% sequence identity to any of these sequences. a heavy chain complementarity determining region (HCDR) 1 having a sequence of SEQ ID NO: 5, 12, 19, 26, 33, 40, 47, 55, 62, 69, 76, 83, 90, 97, 104, 111, 118, 125, or 132, or a sequence having at least 70% sequence identity to any of these sequences; HCDR2 having a sequence with sequence identity to SEQ ID NO: 6, 13, 20, 27, 34, 41, 48, 56, 63, 70, 77, 84, 91, 98, 105, 112, 119, 126, or 133, or a sequence with at least 70% sequence identity to any of these sequences; and HCDR3 having a sequence with SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 57, 64, 71, 78, 85, 92, 99, 106, 113, 120, 127, or 134, or a sequence with at least 70% sequence identity to any of these sequences.

[0044] Also in certain embodiments, the antibody or antibody variant comprises the following combinations of amino acid sequences for LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively: SEQ ID NOs: 2, 3, 4, 5, 6, and 7; or SEQ ID NOs: 9, 10, 11, 12, 13, and 14; or SEQ ID NOs: 16, 17, 18, 19, 20, and 21; or SEQ ID NOs: 23, 24, 25, 26, 27, and 28; or SEQ ID NOs: 30, 31, 32, 33, 34, and 35; or SEQ ID NOs: 37, 38, 39, 40, 41, and 42; or SEQ ID NOs: 44, 45, 46, 47, 48, and 49; or SEQ ID NOs: 52, 53, 54, 55, 56, and 57; or SEQ ID NOs: 59, 60, 61, 62, 63, and 64; or SEQ ID NOs: 66, 67, 68, 69, 70, and 71; or SEQ ID NOs: 73, 74, 75, 76, 77, and 78; or SEQ ID NOs: 80, 81, 82, 83, 84, and 85; or SEQ ID NOs: 87, 88, 89, 90, 91, and 92; or SEQ ID NOs: 94, 95, 96, 97, 98, and 99; or SEQ ID NOs: 101, 102, 103, 104, 105, and 106; or SEQ ID NOs: 108, 109, 110, 111, 112, and 113; or SEQ ID NOs: 115, 116, 117, 118, 119, and 120; or SEQ ID NOs: 122, 123, 124, 125, 126, and 127; or SEQ ID NOs: 129, 130, 131, 132, 133, and 134 Preferably, the paratope is defined by Each sequence herein can be an independently sequence having at least 70% sequence identity with the indicated SEQ ID NO.

[0045] The above sequence identities may be higher, for example selected from at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

[0046] Sequence identity, in this context, is determined by the following parameters: Substitution matrix: EBLOSUM62 Gap penalty: 10.0 Extended penalty: 0.5 The alignment is determined by optimal pairwise global alignment using the Needleman-Wunsch algorithm.

[0047] Some preferred antibodies or antibody variants of the first aspect comprise: - a V having an amino acid sequence identical to or present in an amino acid stretch bounded at its C-terminus to the sequence GGGGSGGGSGGGS (SEQ ID NO: 137) in any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, and 128; H Area, and - V having an amino acid sequence that is present in or identical to an amino acid stretch in any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, and 128 L Region V having an amino acid sequence that is present or identical to the amino acid stretch bounded at the N-terminus to the sequence GGGGGSGGGGSGGGSGGGGS (SEQ ID NO: 137) in the same SEQ ID NO. HArea, or - V having an amino acid sequence that is present or identical to the amino acid stretch bound at its C-terminus to the sequence GGGGSGGGGSGGGGSGGG (SEQ ID NO: 138) in SEQ ID NO: 121 H Area, and - V having an amino acid sequence that is present or identical to the amino acid stretch bound at its N-terminus to the sequence GGGGSGGGGSGGGGSGGG (SEQ ID NO: 138) in the same SEQ ID NO. L region including a combination of Here, V H and V L Each of the sequences can independently have at least 80% sequence similarity or sequence identity with the sequence number shown, which can be higher, such as selected from at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

[0048] In this context, sequence similarity and identity are determined by the following parameters: Substitution matrix: EBLOSUM62 Gap penalty: 10.0 Expansion penalty: 0.5 The alignment is determined by optimal pairwise global alignment using the Needleman-Wunsch algorithm.

[0049] In some embodiments, the CS glycosaminoglycan chains bound by any of the antibodies / variants of the invention have N-acetylgalactosamine (GalNAc) residues with a sulfate group at the C-4 position in more than 50%, such as more than 60%, preferably more than 70% of the disaccharide repeats of the chain. Furthermore, the CS chains may optionally contain 4-O sulfated GalNAc and iduronic acid (IdoA). In other embodiments, the CS glycosaminoglycan chains bound by any of the antibodies / variants of the invention have entirely 6-O sulfated N-acetylgalactosamine (GalNAc) residues, and optionally these CS glycosaminoglycans do not contain 4-O sulfated GalNAc and / or iduronic acid (IdoA).

[0050] As mentioned above, the antibodies and antibody variants of the present invention exhibit unprecedented affinity for oFCS (see Examples). For some applications, preferred antibodies and variants among those disclosed above have equilibrium dissociation constants (K D ) ofCS glycosaminoglycan chains less than 10 nM, e.g., less than 5 nM. However, for other applications, modifications of antibody fragments with KDs of up to 40 nM are preferred, e.g., in a monomeric monovalent format with a high on rate and higher off rate, and K d Compounds with values ​​up to 40 nM and low molecular weights that allow easier penetration into solid tumors or are cleared more rapidly for radiodiagnostic purposes are preferred.

[0051] The antigen of the antibody or antibody variant described above in relation to the first aspect is one in which the ofCS glycosaminoglycan chains are attached to a protein core forming a chondroitin sulfate proteoglycan (CSPG) present on the cell membrane or in secreted form in the extracellular matrix. The CSPG is typically selected from, but is not limited to, any one of brain natriuretic peptide B, endothelial cell specific molecule 1, Sushi repeat containing protein SRPX, decorin, protein AMBP, biglycan, myeloid proteoglycan, syndecan 4, amyloid-like protein 2, HLA class II histocompatibility antigen gamma chain, chondroitin sulfate proteoglycan 4, agrin, testoscan 1-3, neuropilin, CD44 antigen, glypican-1-6, syndecan-1-34, laminin subunit gamma 2, carbonic anhydrase 9, aggrecan, perlecan, collagen alpha-1(XII), collagen alpha-1(XV), collagen alpha-1(XVIII), laminin subunit alpha-4, matrix remodeling associated protein 5, nidogen-2, endocan, and versican.

[0052] As already mentioned above, the exact format of the antibody of the first aspect is not critical, although IgG is preferred for some purposes. With respect to variants, as indicated herein, they can have any of the following non-limiting list of formats: Fab, Fab', Fab-SH, F(ab)2, F(ab')2, ScFv, Fv fragments, heavy chain Ig (such as llama or camel Ig), V, or VI. HH Fragments, dsFV, minibodies, diabodies, triabodies, kappabodies, IgNAR, tandAb, BiTEs, and multispecific antibodies. When the antibody is multispecific, a bispecific format is preferred, such as a bispecific antibody that binds to CD3.

[0053] Second aspect of the invention and embodiments thereof This aspect relates to a conjugate or fusion protein comprising at least a first and a second moiety, wherein the first moiety is an antibody or antibody variant according to the first aspect of the invention or any embodiment thereof disclosed herein, and the second moiety is a molecule or polypeptide, wherein the first moiety is conjugated or genetically fused to the second moiety, and the second moiety provides or improves a therapeutic and / or diagnostic function of the conjugate or fusion protein.

[0054] More specifically, this embodiment provides for the attachment of the antibody / variant to a fusion partner conjugation that can provide various advantages in the detection, purification, or therapeutic activity of the antibody / variant, or provide advantages in the therapeutic activity of the conjugation / fusion partner, since the antibody can act as a targeting agent.

[0055] For example, the second moiety may be selected from any of a toxin or fragment thereof (useful in cancer therapy), an immunomodulatory molecule or fragment thereof (also relevant for cancer therapy), a nanoparticle (which may be useful both as a detection label and as a therapeutic moiety), a radionuclide or radionuclide-containing material (also relevant for both therapeutic and diagnostic applications), and a label (i.e., a detectable moiety). The second moiety may also be a gene therapy agent, such as a polynucleotide or oligonucleotide (e.g., an mRNA, e.g., an mRNA containing capped nucleotides) or a polynucleotide or oligonucleotide containing modified nucleosides. For example, the modification may involve any modification of the pentose moiety, sugar / backbone, backbone, base moiety, as well as the introduction of unnatural base pairs. Thus, the sugar modifications are 2'FRNA, 2'OmeRNA, LNA, FANA, HNA, or 2'MOE; the sugar / backbone modifications are mirrorDNA, ribroNA, TNA, t-PhoNA, or dXNA; the backbone modifications are PS, phNA, PNA, and boranophosphate; the base modifications are C7-modified deazaadenine, C-7-modified deazaguanosine, C5-modified cytosine, and C5-modified uridine; the modifications include the addition of hydrogen, chloride, fluoride, or bromide; and the unnatural base pair UBPs are dZ-dP, Ds-Px, 5SICSN-aM, dS-dB, Ds-Pa, and TPT3-NaM. For more details on such modifications, see Duffy K et al. (2020), BMC Biology 18: 112 (doi.org / 10.1186 / s12915-020-00803-6). The fusion / binding partner may also be an aptamer.

[0056] Also, the second moiety can be, for example, another specific antibody or antibody variant, providing a bispecific or multispecific antibody format. For example, the other moiety can be anti-CD3 fused to the antibody or antibody variant of the invention. Another possibility is to couple the antibody of the invention to a molecule containing a radionuclide. For a review of state-of-the-art radionuclides useful for targeted radionuclide therapy, see Goldsmith SJ (2020), Semin Nucl Med 50(1):87-97. (doi: 10.1053 / j.semnuclmed.2019.07.006), which is incorporated herein by reference in its entirety. Alternatively, such radionuclides may be an integral part of the antibody or antibody variant of the invention instead of being part of the second moiety.

[0057] Preferred radionuclides include, but are not limited to, iodine-131, yttrium-90, lutetium-177 (all of which are commonly used in therapy), as well as fluorine-18, gallium-67, krypton-81m, rubidium-82, nitrogen-13, technetium-99m, indium-111, iodine-123, xenon-133, and thallium-201 (all of which are commonly used in imaging techniques).

[0058] In an important embodiment, the second part is a toxin, which can be very useful in treatment.When the antibody targets only or at least preferentially targets tumor tissue, such toxin can be any type that induces cell death (necrotic cell death, or preferably apoptotic cell death), but in most embodiments, it is preferred to be selected from cytotoxic or cytostatic agents, such as alkylating agents, antimetabolites, anti-microtubule agents such as monomethyl auristatin E (MMAE), topoisomerase inhibitors such as exetecan, and cytotoxic antibiotics.

[0059] Essentially, any cytotoxic or cytostatic agent commonly used in cancer therapy can be used, however, conjugation with the antibody / variant expands the selection of cytotoxic and cytostatic agents to those that are too toxic for systemic administration (MMAE is one example), but can be used because the conjugation with the antibody / variant prevents systemic diffusion of the toxin.

[0060] Alkylating agents include nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatin and derivatives. Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan. Nitrosoureas include N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine and streptozotocin. Tetrazines include dacarbazine, mitozolomide and temozolomide. Aziridines include thiotepa, mitomycin and diaziquone (AZQ). Cisplatin and derivatives include cisplatin, carboplatin and oxaliplatin. Additionally, alkylating agents also include procarbazine and hexamethylmelamine.

[0061] Antimetabolites include antifolates, fluoropyrimidines, deoxynucleoside analogs, and thiopurines. Antifolates include methotrexate and pemetrexed. Fluoropyrimidines include fluorouracil and capecitabine. Deoxynucleoside analogs include cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, and pentostatin. Thiopurines include thioguanine and mercaptopurine.

[0062] Microtubule inhibitors include the vinca alkaloids and taxanes, where the vinca alkaloids include vincristine, vinblastine, vinorelbine, vindesine, and vinflunine. The taxanes include paclitaxel, docetaxel.

[0063] Podophyllotoxin is also an anti-microtubule agent and exhibits similar effects to the vinca alkaloids.

[0064] Topoisomerase inhibitors include irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, camptothecin, and aclarubicin.

[0065] Cytotoxic antibiotics include anthracyclines, bleomycin, mitomycin C, and actinomycin. Important anthracyclines are doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone. Immune checkpoint inhibitors include those that target CTLA4, PD-1, or PD-L1, and include ipilimumab (targeting CTLA-4), nivolumab (targeting PD-1), pembrolizumab (targeting PD-1), atezolizumab (targeting PDL-1), avelumab (targeting PDL-1), durvalumab (targeting PDL-1), and cemiplimab (targeting PD-1). In addition, inhibitors of actively expressing ubiquitin ligases, such as CISH (cytokine-inducible SH2-containing protein) and CBL, are also relevant.

[0066] Some conjugates of the second embodiment are those in which the second moiety is a polypeptide and the first moiety is genetically fused to the second moiety. Particularly interesting fusion partners for antibodies / variants are cytokines and chemokines, of particular importance interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 12, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL).

[0067] The third aspect of the invention and its embodiments This aspect relates to a polypeptide comprising i) a first polypeptide domain which is a transmembrane domain and an endodomain of a chimeric antigen receptor (CAR), and ii) a second polypeptide domain which is an antibody or antibody variant according to the first aspect of the invention or any embodiment thereof disclosed herein. Such a fusion protein is particularly relevant for the production of therapeutically useful CAR-T cells.

[0068] Details regarding the construction of antibody-CAR fusions are described, for example, in Larson, RC et al. (2021), Nature Reviews Cancer 21: 145-161 and Feins S et al. (2019), Hematol 94(S1): S3-S9.

[0069] The fourth aspect of the invention and its embodiments This aspect relates to a CAR-T cell comprising a polypeptide of the third aspect of the invention or any embodiment thereof disclosed herein.

[0070] Details on the construction of CAR-T cells can be found, for example, in Larson, RC et al. (2021), Nature Reviews Cancer 21: 145-161 and Feins S et al. (2019), Hematol 94(S1): S3-S9.

[0071] Fifth aspect of the invention and embodiments thereof This aspect relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or antibody variant of the first aspect of the invention or an embodiment disclosed herein, a conjugate or fusion protein of the second aspect of the invention or an embodiment disclosed herein, or a polypeptide of the third aspect of the invention or an embodiment disclosed herein.

[0072] The antibodies / variants disclosed herein are primarily derived from human B cells, meaning that the genetic material associated with antibody production in recombinant systems is suitable for expression in human cells. However, codons can be altered according to the genetic code, thereby optimizing expression for recombinant production in virtually any host cell.

[0073] Moreover, one skilled in the art can introduce the requisite antigen binding regions (Fv or CDRs) into any vector backbone that encodes the desired antibody or antibody variant format.

[0074] The sixth aspect of the present invention and its embodiments This aspect relates to a vector, such as an expression vector or a cloning vector, comprising the isolated nucleic acid molecule of the fifth aspect of the invention or any embodiment thereof disclosed herein. If the vector is an expression vector, the vector contains the genetic elements necessary for this purpose:

[0075] One preferred vector disclosed herein comprises, in operable linkage and in a 5'-3' orientation, an expression control region comprising an enhancer / promoter for driving expression of the nucleic acid fragment of the fifth aspect, optionally a signal peptide coding sequence, a nucleotide sequence of the fifth aspect, and optionally a terminator. Such a vector thus constitutes an expression vector useful for intracellular production of the antibody, variant or polypeptide of the first aspect, and, where relevant, the second and third aspects (collectively referred to as "polypeptides of the invention"). Since the polypeptides of the invention are of mammalian origin, recombinant production is conveniently carried out in eukaryotic host cells, and thus it is preferred here that the expression control region drives expression in eukaryotic cells (such as plant, fungal, insect and mammalian cells). However, since many antibody variants can be produced in bacteria, it is preferred here that the expression control region drives expression in eukaryotic cells, prokaryotic cells such as bacteria, for example E. coli.

[0076] The vectors may further comprise a sequence encoding a signal peptide, as indicated, which allows for secretion of the expression product from the vector or integration into a membrane.

[0077] In any event, certain of the vectors disclosed herein are capable of autonomous replication.

[0078] The vectors disclosed herein may also be vectors that can be integrated into the genome of a host cell. This is particularly useful when the vector is used to generate a stably transformed cell, in which case the progeny also contain the genetic information introduced via the vector. Alternatively, vectors that cannot be integrated into the genome of a mammalian host cell are useful for initial screening of expression efficiency.

[0079] Typically, the vectors disclosed herein are selected from the group consisting of viruses, bacteriophages, plasmids, minichromosomes, and cosmids.

[0080] A more detailed description of the vectors disclosed herein is provided below:

[0081] The polypeptides disclosed herein may be encoded by the nucleic acid of the fifth aspect contained in a vector. The nucleic acid sequence may be "heterologous", meaning that it is in a context foreign to the cell into which the vector is introduced, including sequences that are homologous to sequences in the cell, but in a location not normally found in the host cell. Vectors include naked DNA, RNA, plasmids, cosmids, viruses (bacteriophages, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). Those skilled in the art are well equipped to construct vectors by standard recombinant techniques (e.g., Sambrook et al, 2001; Ausubel et al, 1996, both incorporated herein by reference). In addition to encoding the polypeptides of the present invention, the vectors of the present invention may also encode polypeptide sequences such as tags or fusion partners that stimulate the immune system, such as cytokines or active fragments thereof. Useful vectors encoding such fusion proteins include pIN vectors, vectors encoding stretches of histidine residues, and pGEX vectors, which are used to generate glutathione S-transferase (GST) soluble fusion proteins for subsequent purification and isolation or cleavage.

[0082] The vectors disclosed herein may be used in host cells to produce the polypeptides disclosed herein, which may then be purified for administration to a subject.

[0083] Expression vectors can contain a variety of "control sequences," which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that control transcription and translation, vectors and expression vectors can contain nucleic acid sequences that serve other functions as well, and are described below.

[0084] 1. Promoters and Enhancers A "promoter" is a regulatory sequence. A promoter is usually a region of a nucleic acid sequence where the initiation and rate of transcription are controlled. A promoter may contain genetic elements to which regulatory proteins or molecules, such as RNA polymerase or other transcription factors, may bind. The phrases "operably positioned," "operably linked," "under control," and "under transcriptional control" mean that the promoter is in the correct functional location and / or orientation relative to a nucleic acid sequence and controls the transcription initiation and expression of that sequence. A promoter may or may not be used in conjunction with an "enhancer," which refers to a cis-acting regulatory sequence involved in the transcriptional activation of a nucleic acid sequence.

[0085] A promoter may be a promoter naturally associated with a gene or sequence, obtained by isolating 5' non-coding sequences upstream of a coding segment or exon. Such promoters are sometimes referred to as "endogenous". Similarly, an enhancer may be an enhancer naturally associated with a nucleic acid sequence and located downstream or upstream of that sequence. Alternatively, certain advantages are obtained by placing a coding nucleic acid segment under the control of a recombinant or heterologous promoter. A recombinant or heterologous promoter refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. A recombinant or heterologous enhancer also refers to an enhancer that is not normally associated with a nucleic acid sequence in its natural state. Such promoters or enhancers include promoters or enhancers of other genes, and promoters or enhancers isolated from other prokaryotic, viral, or eukaryotic cells, and "non-naturally occurring" promoters or enhancers, i.e., promoters or enhancers that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences can also be produced using recombinant cloning and / or nucleic acid amplification techniques, including PCR®, in connection with the compositions disclosed herein.

[0086] Of course, it may be important to use a promoter and / or enhancer that effectively induces the expression of the DNA segment in the cell type or organism selected for expression. Those skilled in the field of molecular biology are generally aware of the use of promoter, enhancer, and cell type combinations for protein expression (see Sambrook et al, 2001, incorporated herein by reference). The promoter used may be constitutive, tissue-specific, or inducible, and in certain embodiments, may induce high-level expression of the introduced DNA segment under certain conditions, such as large-scale production of recombinant protein or peptide.

[0087] Examples of inducible elements are regions of nucleic acid sequences that are activated in response to a particular stimulus, such as immunoglobulin heavy chains, immunoglobulin light chains, T cell receptors, HLA DQα and / or DQβ, β-interferon, interleukin-2, interleukin-2 receptor, MHC class II5, MHC class II HLA-DRα, β-actin, muscle creatine kinase (MCK), prealbumin (transthyretin), elastase I, metallothionein (MTII), collagenase, albumin, α-fetoprotein, γ-globin, β-globin, c-fos, c-HA-ras, These include, but are not limited to, insulin, neural cell adhesion molecule (NCAM), alphal-antitrypsin, H2B (TH2B) histone, mouse and / or type I collagen, glucose-regulated proteins (GRP94 and GRP78), rat growth hormone, human serum amyloid A (SAA), troponin I (TNI), platelet-derived growth factor (PDGF), Duchenne muscular dystrophy, SV40, polyoma, retrovirus, papilloma virus, hepatitis B virus, human immunodeficiency virus, cytomegalovirus (CMV) IE, and gibbon ape leukemia virus.

[0088] Inducible elements include MTII-phorbol ester (TFA) / heavy metals, MMTV (mouse mammary tumor virus)-glucocorticoids; beta-interferon-poly(rl)x / poly(rc); adenovirus 5E2-ElA; collagenase-phorbol ester (TPA); stromelysin-phorbol ester (TPA); SV40-phorbol ester (TPA); mouse MX gene-interferon, Newcastle disease virus; GRP78 gene-A23187; alpha-2-macroglobulin-IL-6; vimentin-serum; MHC class I gene H-2kappa b-interferon; HSP70-E1A / SV40 large T antigen; proliferin-phorbol ester / TPA; tumor necrosis factor-PMA; and thyroid stimulating hormone alpha gene-thyroid hormone.

[0089] The Dectin-1 and Dectin-2 promoters are also believed to be useful in the present invention. Additionally, any promoter / enhancer combination (according to the Eukaryotic Promoter Database EPDB) can be used to drive expression of structural genes encoding oligosaccharide processing enzymes, protein folding assisting proteins, selectable marker proteins, or heterologous proteins of interest.

[0090] The specific promoter used to control the expression of the polynucleotide encoding the peptide or protein disclosed herein is not considered to be important, as long as it can express the polynucleotide in target cells, preferably bacterial cells.When human cells are the target, it is preferred to place the polynucleotide coding region adjacent to and under the control of a promoter that can be expressed in human cells.Generally speaking, such promoters may include bacterial, human, or viral promoters.

[0091] In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, and the Rous sarcoma virus long terminal repeat can be used to obtain high level expression of the polynucleotides associated with the present invention. The use of other viral or mammalian cell or bacterial phage promoters well known in the art to achieve expression of the polynucleotides is also contemplated.

[0092] In the embodiment in which the vector is administered to the subject for protein expression, it is considered that the promoter that is desirable for use with the vector is a promoter that is not downregulated by cytokines, or even if downregulated, is strong enough to produce a sufficient amount of the protein / polypeptide of the present invention to induce an immune response in the subject.Non-limiting examples of these include CMV IE and RSV LTR.In other embodiments, a promoter that is upregulated in the presence of cytokines is used.The MHCI promoter increases expression in the presence of IFN-γ.

[0093] Tissue-specific promoters can be used, especially when the antigen is to be expressed in cells in which it is desired, such as dendritic cells or macrophages. The mammalian MHC I and MHC II promoters are examples of such tissue-specific promoters. 2. Initiation signals and internal ribosome binding sites (IRES)

[0094] Specific initiation signals may also be required for efficient translation of the coding sequence. These signals include the ATG start codon or adjacent sequences. Exogenous translational control signals, including the ATG start codon, may need to be provided. One of ordinary skill in the art can readily determine this and provide the necessary signals. It is well known that to ensure translation of the entire insert, the initiation codon must be "in frame" with the reading frame of the desired coding sequence. The exogenous translational control signals and initiation codons can be either natural or synthetic and can be operable in bacterial or mammalian cells. The efficiency of expression can be increased by including appropriate transcriptional enhancer elements.

[0095] In certain embodiments disclosed herein, the use of an internal ribosome entry site (IRES) element is for creating multigene or polycistronic messages. The IRES element can circumvent the ribosome scanning model of 5' methylated cap-dependent translation and initiate translation at an internal site. IRES elements from two members of the picornavirus family (polio and encephalomyocarditis) as well as IRES from mammalian messages have been described. The IRES element can link heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating a polycistronic message. Thanks to the IRES element, each open reading frame is accessible to ribosomes for efficient translation. A single promoter / enhancer can be used to efficiently express multiple genes and transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, which are incorporated herein by reference).

[0096] 2. Multiple cloning site A vector may contain a multiple cloning site (MCS), which is a nucleic acid region that contains multiple restriction enzyme sites, any of which can be used to digest the vector in conjunction with standard recombinant techniques. Often, vectors are linearized or fragmented using a restriction enzyme that cuts within the MCS, allowing foreign sequences to be ligated into the vector. Techniques involving restriction enzymes and ligation reactions are well known to those skilled in the art of recombinant technology.

[0097] 3. Splicing Sites Most transcribed eukaryotic RNA molecules undergo RNA splicing to remove introns from the primary transcript. As is relevant in the context of the vectors of the present invention, vectors containing eukaryotic genomic sequences may require donor and / or acceptor splicing sites to ensure proper processing of the transcript for protein expression.

[0098] 4. Termination signal The vector or construct of the present invention usually comprises at least one termination signal. "Termination signal" or "terminator" is composed of the DNA sequence that is involved in the specific termination of RNA transcript by RNA polymerase.Therefore, in certain embodiments, the termination signal that ends the production of RNA transcript is considered.Terminator may be necessary to achieve desired message level in vivo.

[0099] In eukaryotic systems, the terminator region may also contain specific DNA sequences that allow site-specific cleavage of the new transcript to expose a polyadenylation site. This signals a specialized endogenous polymerase to add a stretch of about 200 A residues (polyA) to the 3' end of the transcript. RNA molecules modified with this polyA tail appear to be more stable and are translated more efficiently. Thus, in other embodiments involving eukaryotes, it is preferred that the terminator contains a signal for cleavage of the RNA, and more preferably that the terminator signal promotes polyadenylation of the message.

[0100] Terminators contemplated for use in the present invention include known terminators of transcription described herein or known to those of skill in the art, including, but not limited to, for example, the bovine growth hormone terminator or viral termination sequences such as the SV40 terminator. In certain embodiments, the termination signal may be the absence of a transcribable or translatable sequence, such as by truncation of the sequence.

[0101] 5. Polyadenylation Signal In expression, particularly eukaryotic expression (relevant to nucleic acid vaccination), a polyadenylation signal is usually included to ensure proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be critical to the successful implementation of the present invention, and / or any such sequence can be used. A preferred embodiment includes the SV40 polyadenylation signal and / or the bovine growth hormone polyadenylation signal, which are known to be convenient and / or function well in various target cells. Polyadenylation may increase the stability of the transcript or facilitate cytoplasmic transport. Therefore, the corresponding coding RNA fragment preferably includes a poly(A) tail.

[0102] 6. Origin of replication To propagate a vector in a host cell, the vector can contain one or more origin of replication sites (often referred to as "ons"), which are specific nucleic acid sequences at which replication is initiated. Alternatively, if the host cell is yeast, an autonomously replicating sequence (ARS) can be used.

[0103] 7. Selectable and Screenable Markers In certain embodiments disclosed herein, cells containing the nucleic acid construct of the present invention can be identified in vitro or in vivo by encoding a screenable or selectable marker in the expression vector. The marker, when transcribed and translated, confers a distinguishable change to the cell, allowing cells containing the expression vector to be easily identified. Generally, a selectable marker confers a property that allows selection. A positive selection marker is one whose presence allows selection, and a negative selection marker is one whose presence prevents selection. Examples of positive selection markers include drug resistance markers.

[0104] Typically, the inclusion of a drug selection marker facilitates cloning and identification of transformants. For example, markers that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, and histidinol are useful selection markers. In addition to markers that confer a phenotype that allows the transformants to be distinguished based on the implementation of conditions, other types of markers include screenable markers such as GFP for colorimetric analysis. Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) and chloramphenicol acetyltransferase (CAT) can be utilized. Those skilled in the art will also know how to use immune markers that can be used in conjunction with FACS analysis. The marker used is not believed to be important, so long as it can be expressed simultaneously with the nucleic acid encoding the protein disclosed herein. Further examples of selectable and screenable markers will be familiar to those skilled in the art.

[0105] The seventh aspect of the invention and its embodiments This aspect relates to a host cell comprising or transformed with the vector of the sixth aspect of the invention or any embodiment thereof disclosed herein.

[0106] The transformed cells disclosed herein are useful as organisms for producing the polypeptides or chimeric polypeptides of the present invention, but are also useful as simple "containers" for the nucleic acids and vectors disclosed herein.

[0107] Certain transformed cells disclosed herein are capable of replicating the nucleic acid fragment of the fifth aspect. Preferred transformed cells disclosed herein are capable of expressing the nucleic acid of the fifth aspect.

[0108] For recombinant production, it is convenient but not essential that the transformed cell be a prokaryote such as a bacterium, although in general both prokaryotic and eukaryotic cells can be used, see considerations above.

[0109] Suitable prokaryotic cells are bacterial cells selected from the group consisting of Escherichia coli (such as E. coli), Bacillus (such as Bacillus subtilis), and Salmonella. Preferred E. coli cells are shuffle cells or similar E. coli strains that form disulfide bonds in the cytoplasm to allow scFV folding.

[0110] Eukaryotic cells can be in the form of yeast (such as Saccharomyces cerevisiae) and protozoa. Alternatively, the transformed eukaryotic cells can be derived from multicellular organisms such as fungi, insect cells, plant cells, or mammalian cells, such as human cells.

[0111] For production purposes, it is advantageous that the transformed cell disclosed herein is stably transformed by stably integrating the nucleic acid defined above for option i) into its genome, and in certain embodiments it is also preferred that the transformed cell secretes or carries on its surface the polypeptide disclosed herein, as this facilitates recovery of the produced polypeptide. A particular version of this embodiment is one in which the transformed cell is a bacterium and the polypeptide disclosed herein is secreted into the periplasmic space.

[0112] An interesting production system is the use of plants. For example, proteins can be produced in plants at low cost by genetically modifying the plants using the Agrobacterium transfection system to express a gene encoding the protein of interest. Commercially available platforms are provided by iBio CMO LLC (8800 HSC Pkwy, Bryan, TX 77807, USA) and iBio, Inc (9 Innovation Way, Suite 100, Newark, DE 19711, USA) and are disclosed, for example, in EP2 853 599, EP1 769 068, and EP2 192 172. Thus, in such systems, the vector is an Agrobacterium vector or other vector suitable for transfection of plants.

[0113] As mentioned above, stably transformed cells are preferred, i.e. this allows the establishment of cell lines consisting of transformed cells as defined herein, such cell lines being a particularly preferred aspect of the present invention.

[0114] Further details regarding cells and cell lines are provided below:

[0115] The cell suitable for recombinant nucleic acid expression of the nucleic acid fragment of the present invention is prokaryotic and eukaryotic.Examples of prokaryotic cells include E.coli Shuffle; Staphylococcus genus member, such as S.epidermidis; Lactobacillus genus member, such as L.plantarum; Lactococcus genus member, such as L.lactis; Bacillus genus member, such as B.subtilis; Corynebacterium genus member, such as C.glutamicum; and Pseudomonas genus member, such as Ps.fluorescens. Examples of eukaryotic cells include mammalian cells; insect cells; yeast cells, such as Saccharomyces (e.g., S. cerevisiae), Pichia (e.g., P. pastoris), Hansenula (e.g., H. polymorpha), Kluyveromyces (e.g., K. lactis or K. fragilis), and Schizosaccharomyces (e.g., S. pombe). As mentioned above, the nucleic acid sequences of the present invention can be appropriately codon-optimized to facilitate efficient expression from each of the transformed cells disclosed herein.

[0116] Techniques for producing recombinant genes, introducing them into cells, and expressing them are well known in the art. Examples of such techniques are described in references such as Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-2002, and Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989.

[0117] As used herein, the terms "cell," "cell line," and "cell culture" may be used interchangeably. All of these terms include their progeny, i.e., all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing heterologous nucleic acid sequences, "host cell" refers to a prokaryotic or eukaryotic cell, including any transformable organism capable of replicating a vector or expressing a heterologous gene encoded by the vector. Host cells can be, and have been, used as recipients for vectors and viruses. Host cells may be "transfected" or "transformed," which refers to the process by which exogenous nucleic acid, such as a recombinant protein-coding sequence, is transferred or introduced into a host cell. A transformed cell includes the primary subject cell and its progeny.

[0118] Host cells may be obtained from prokaryotic or eukaryotic organisms, such as bacteria, yeast cells, insect cells, or mammalian cells, for replication of the vector or expression of part or all of the nucleic acid sequence. Numerous cell lines and cultures can be used as host cells, which are available from other depositories, such as the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSM), organizations that serve as archives of living cultures and genetic material. An appropriate host can be determined by one of skill in the art based on the backbone of the vector and the desired outcome. For example, plasmids or cosmids can be introduced into prokaryotic host cells for replication of many vectors or expression of the encoded proteins. Bacterial cells used as host cells for replication and / or expression of vectors include Staphylococcus aureus strains, DH5α, JM109, and KC8, as well as a number of commercially available bacterial hosts, such as SURE® Competent Cells and SOLOP ACK™ Gold Cells (STRATAGENE®, La Jolla, CA). Alternatively, bacterial cells, such as E. coli LE392, can be used as host cells for phage viruses. Suitable yeast cells include Saccharomyces cerevisiae, Saccharomyces pombe, and Pichia pastoris.

[0119] Examples of eukaryotic host cells for replicating and / or expressing vectors include HeLa, NIH3T3, Jurkat, 293, Cos, CHO, Saos, HEK293 and PC12. Many host cells from various cell types and organisms are available and known to those skilled in the art. Similarly, viral vectors can be used in combination with eukaryotic or prokaryotic host cells, particularly host cells that are permissive for replicating or expressing vectors.

[0120] Some vectors may use control sequences that allow them to replicate and / or express in both prokaryotic and eukaryotic cells. Those skilled in the art will further understand the conditions under which all of the above host cells are maintained and incubated to allow vector replication. Also understood and known are the techniques and conditions that allow large-scale production of vectors, and the production of nucleic acids encoded by the vectors and their cognate polypeptides, proteins, or peptides.

[0121] There are numerous expression systems that contain at least some or all of the above-mentioned components. In the present invention, prokaryotic and / or eukaryotic based systems can be used to produce nucleic acid sequences, or their cognate polypeptides, proteins, and peptides. Many such systems are commercially available and widely available.

[0122] Insect cell / baculovirus systems can produce high level protein expression of heterologous nucleic acid segments as described in U.S. Pat. Nos. 5,871,986 and 4,879,236 (both of which are incorporated herein by reference) and can be purchased commercially, for example, from INVITROGEN® under the name MAXBAC® 2.0 and from CLONTECH® as BACPACK® Baculovirus Expression System.

[0123] In addition to the expression systems disclosed herein, other examples of expression systems include STRATAGENE®'s COMPLETE CONTROL® Inducible Mammalian Expression System, which contains a synthetic ecdysone-inducible receptor, or its E. coli expression system, the pET Expression System. Another example of an inducible expression system is available from INVITROGEN® with their T-REX® (Tetracycline-Controlled Expression) System, an inducible mammalian expression system that uses a full-length CMV promoter. INVITROGEN® also offers a yeast expression system called the Pichiaethanolica Expression System, which is designed to produce high levels of recombinant proteins in the methylotrophic yeast Pichiaethanolica. One of skill in the art would know how to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.

[0124] Eighth aspect of the present invention and embodiments thereof This aspect relates to a method for producing an antibody or antibody variant according to the first aspect of the invention or an embodiment disclosed herein, a conjugate or fusion protein according to the second aspect of the invention or an embodiment disclosed herein, or a polypeptide according to the third aspect of the invention or an embodiment disclosed herein, the method comprising the steps of transfecting or transforming a host cell with a vector according to the sixth aspect of the invention or an embodiment disclosed herein, expressing a nucleotide sequence according to the fifth aspect of the invention or an embodiment disclosed herein, and isolating the antibody or antibody variant, conjugate or fusion protein, or polypeptide.

[0125] In general, the disclosure provided above under the sixth and seventh aspects also applies mutatis mutandis to the eighth aspect.

[0126] Ninth aspect of the invention and embodiments thereof This aspect relates to a pharmaceutical composition comprising an antibody or antibody variant of the first aspect of the invention or an embodiment disclosed herein, a conjugate or fusion protein of the second aspect of the invention or an embodiment disclosed herein, or a CAR-T cell of the fourth aspect of the invention or an embodiment disclosed herein, and a pharma- ceutically acceptable carrier, vehicle or diluent.

[0127] Thus, this aspect generally relates to pharmaceutical compositions comprising the antibody or variants thereof. For details of such formulations, including selected carriers, vehicles, diluents and excipients, reference is generally made to the state of the art for formulating antibodies and their derivatives, see below.

[0128] In some embodiments, a pharmaceutical composition can contain at least 0.1% by weight of the antibody / antibody variant, e.g., at least 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the antibody / antibody variant. In other embodiments, for example, the antibody / antibody variant can comprise about 2% to about 75%, about 25% to about 60%, about 30% to about 50% by weight of the composition, or any range therein.

[0129] The pharmaceutical composition further comprises one or more additional components. A pharma- ceutically acceptable carrier can be a carrier approved by a federal or state regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, particularly humans.

[0130] The preparation of pharmaceutical compositions which contain an antibody or other molecule described herein as an active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed., 1990, incorporated herein by reference. Moreover, it will be understood that for animal (including human) administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards or the European Medicines Agency (EMA).

[0131] Pharmaceutically acceptable carriers include liquid, semi-solid, i.e., paste, or solid carriers. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, and the like, or combinations thereof. Pharmaceutically acceptable carriers include aqueous vehicles (e.g., water, alcoholic / aqueous solutions, ethanol, saline, parenteral vehicles such as sodium chloride, Ringer's dextrose, and the like), non-aqueous vehicles (e.g., propylene glycol, polyethylene glycol, vegetable oils, and injectable organic esters such as ethyl oleate), dispersion media, coatings (e.g., lecithin), surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents, inert gases, parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal), isotonic agents (e.g., sugars, sodium chloride), absorption delaying agents (e.g., aluminum monostearate, gelatin), salts, drugs, drug stabilizers (e.g., buffers, amino acids such as glycine and lysine, dextrose, and the like, as known to those skilled in the art). The composition may include materials such as carbohydrates, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavors, dyes, liquids and nutritional supplements, and combinations thereof, such as sucrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc. The use of conventional vehicles, agents, diluents, or carriers in administrable compositions for use in carrying out the methods is appropriate, so long as they are not deleterious to the recipient or to the therapeutic effect of the composition contained therein. The pH and exact concentration of the various components in the pharmaceutical composition are adjusted according to well-known parameters. According to certain aspects of the present disclosure, the composition may be combined with the carrier by any convenient and practical method, i.e., dissolving, suspending, emulsifying, mixing, encapsulating, absorbing, pulverizing, etc. Such procedures are routine for those skilled in the art.

[0132] In some embodiments, the pharma-ceutically acceptable carrier can be an aqueous pH buffered solution. Examples include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; low molecular weight (e.g., less than about 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0133] In some embodiments, the pharma- ceutically acceptable carrier can be a sterile liquid, such as water or an oil, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water can be a carrier, particularly when the pharmaceutical composition is administered intravenously. Saline, aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, nonfat dry milk, glycerol, propylene, glycol, water, ethanol, polysorbate 80. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like.

[0134] Certain embodiments of the present disclosure can have different types of carriers depending on whether they are administered in solid, liquid, or aerosol form and whether they need to be sterile for routes of administration such as injection. The compositions can be formulated for administration intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intramuscularly, subcutaneously, intramucosally, intratumorally, peritumorally, orally, topically, locally, by inhalation (e.g., aerosol inhalation), by injection, by infusion, by continuous infusion, by localized perfusion to directly bathe the target cells, via catheter, via lavage fluid, by administration in lipid compositions (e.g., liposomes), or by other methods known to those skilled in the art or any combination of the aforementioned methods (see, e.g., Remington's Pharmaceutical Sciences, 18th Ed., 1990, which is incorporated herein by reference). Typically, such compositions can be prepared as liquid solutions or suspensions. Solid forms suitable for the addition of liquid to prepare a solution or suspension prior to injection can also be prepared. The formulations can also be emulsified.

[0135] The antibody / antibody variant can be formulated in the composition in free base, neutral, or salt form. Pharmaceutically acceptable salts include acid addition salts, for example salts formed with the free amino groups of the protein composition, or salts formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups can also be derived from inorganic bases, such as sodium, potassium, ammonium, calcium, or iron(III) hydroxides, or organic bases, such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, or procaine.

[0136] In a further embodiment, a pharmaceutical composition comprising lipid is provided. Lipids broadly include a class of substances that have the property of being insoluble in water and extractable with organic solvents. Examples include compounds that contain long-chain aliphatic hydrocarbons and their derivatives. Lipids can be naturally occurring or synthetic (i.e., designed or manufactured by man). Lipids can be biological substances. Biological lipids are well known in the art and include, for example, neutral lipids, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulfatides, lipids containing ether-linked and ester-linked fatty acids, polymerizable lipids, and combinations thereof. Compounds that are understood by those skilled in the art as lipids other than those specifically described herein can also be used.

[0137] Those skilled in the art are familiar with the various techniques that can be used to disperse compositions in lipid vehicles.For example, antibodies can be dispersed in a solution that contains lipids, dissolved with lipids, emulsified with lipids, mixed with lipids, combined with lipids, covalently bound to lipids, contained as a suspension in lipids, contained with micelles or liposomes, or complexed, or bound to lipids or lipid structures by any means known to those skilled in the art.Dispersion may or may not result in the formation of liposomes.

[0138] Generally, the components of the composition are supplied in unit dosage form, either separately or mixed together, for example as a dry lyophilized powder or water-free concentrate in a sealed container, such as an ampoule or sachet indicating the quantity of active agent. When the composition is administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0139] The amount of active ingredient in each therapeutically useful composition can be prepared in such a way that any unit dose of compound can obtain a suitable dosage.Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life and other pharmacological considerations can be taken into account by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and treatment regimens may be desirable.

[0140] "Unit dose" or "unit dosage amount" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined amount of pharmaceutical composition calculated to produce the desired response discussed above in connection with its administration, i.e., the appropriate route and treatment regimen. The amount administered depends on the desired effect, both according to the number of treatments and the unit dose. The actual dosage of the composition of the present embodiment administered to a patient or subject can be determined by physical and physiological factors such as body weight, age, health and sex of the subject, type of disease being treated, degree of disease penetration, previous or concurrent therapeutic interventions, idiopathic nature of the patient, route of administration, and the potency, stability and toxicity of the particular therapeutic agent. In other non-limiting examples, dosages can be from about 1 μg / kg / body weight, about 5 μg / kg / body weight, about 10 μg / kg / body weight, about 50 μg / kg / body weight, about 100 μg / kg / body weight, about 200 μg / kg / body weight, about 350 μg / kg / body weight, about 500 μg / kg / body weight, about 1 mg / kg / body weight, about 5 mg / kg / body weight, about 10 mg / kg / body weight, about 50 mg / kg / body weight, about 100 mg / kg / body weight, about 200 mg / kg / body weight, about 350 mg / kg / body weight, about 500 mg / kg / body weight to about 1000 mg / kg / body weight or more per administration, and any range derivable therein. As non-limiting examples of ranges derived from the numerical values ​​recited herein, based on the numerical values ​​above, ranges can be administered, such as about 5 mg / kg / body weight to about 100 mg / kg / body weight, about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. In any event, the physician responsible for administration will determine the concentration of active ingredient in the composition and the appropriate dosage for the individual subject.

[0141] As will be appreciated by those skilled in the art, the compositions described herein are not limited by the particular nature of the therapeutic formulation. For example, such compositions can be provided as formulations with physiologically acceptable liquid, gel, or solid carriers, diluents, and excipients. These therapeutic formulations, like other therapeutic agents, can be administered to mammals for veterinary use, such as livestock, and for clinical use in humans. In general, the dosage required for therapeutic effect will vary depending on the type of use and method of administration, and the specific requirements of the individual subject. The actual dosage of the composition administered to an animal patient, including a human patient, can be determined by physical and physiological factors, such as body weight, severity of symptoms, type of disease being treated, previous or current therapeutic interventions, idiopathic nature of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or frequency of administration of an effective amount may vary depending on the subject's response. In any event, the administering physician will determine the concentration of active ingredient in the composition and the appropriate dosage for the individual subject.

[0142] A tenth aspect of the present invention and its embodiments This aspect relates to the use of an antibody or antibody variant of the first aspect of the invention or an embodiment disclosed herein, or a conjugate or fusion protein of the second aspect of the invention or an embodiment disclosed herein, for in vitro detection and / or isolation of cancer cells from a subject, such as circulating tumor cells, and for minimal residual disease detection in haematological tumors.

[0143] In this context, reference is made to the detailed disclosure in the Examples and to the context of the fourteenth and fifteenth aspects of the invention and any embodiments thereof disclosed herein.

[0144] However, generally, the cancer cell is derived from a cancer selected from epithelial tumor, non-epithelial tumor (such as blood cancer), and mixed tumor.Epithelial tumor can be either carcinoma or adenocarcinoma, and non-epithelial tumor or mixed tumor can be either liposarcoma, fibrosarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, glioma, neuroblastoma, medulloblastoma, malignant melanoma, malignant meningioma, neurofibrosarcoma, leukemia, myeloproliferative disease, lymphoma (such as B-cell or T-cell lymphoma), angiosarcoma, Kaposi's sarcoma, malignant teratoma, dysgerminoma, seminoma, mesothelioma, or chondrosarcoma melanoma. Also, the anatomical site of cancer can vary: it can be cancer of the lungs, eyes, nose, mouth, tongue, pharynx, esophagus, stomach, colon, rectum, bladder, ureters, urethra, kidneys, liver, pancreas, thyroid, adrenal glands, breast, skin, central nervous system, peripheral nervous system, meninges, vasculature, testes, prostate, ovaries, uterus, cervix, spleen, bone, or cartilage.

[0145] An eleventh aspect of the present invention and its embodiments This aspect is for the use of the antibody or antibody variant of the first aspect of the invention or any embodiment thereof, the conjugate or fusion protein of the second aspect of the invention or any embodiment thereof, the CAR-T cell of the fourth aspect of the invention or any embodiment thereof, or the pharmaceutical composition of the ninth aspect of the invention or any embodiment thereof, as a medicament.

[0146] As will be evident from the following examples, the antibodies and antibody variants of the present invention target a variety of malignancies, making the antibodies and antibody variants and products obtained therefrom highly important in methods for treating malignancies. Typically, the agents are used to treat mammals, particularly humans, but also domestic animals such as dogs, cats and horses.

[0147] A twelfth aspect of the present invention and its embodiments This aspect relates to the use of an antibody or antibody variant of the first aspect of the invention or any embodiment thereof, a conjugate or fusion protein of the second aspect of the invention or any embodiment thereof, or a pharmaceutical composition of the ninth aspect of the invention or any embodiment thereof, for the diagnosis of cancer in a subject.

[0148] As with the therapeutic aspects of the invention, the subjects diagnosed in accordance with the present invention will primarily be mammals, particularly humans, but also domestic animals such as dogs, cats and horses, although reference is made to the more detailed disclosure below regarding diagnostic methods.

[0149] A thirteenth aspect of the present invention and its embodiments This aspect relates to an antibody or antibody variant of the first aspect of the invention or an embodiment disclosed herein, a conjugate or fusion protein of the second aspect of the invention or an embodiment disclosed herein, a CAR-T cell of the fourth aspect of the invention or an embodiment disclosed herein, or a pharmaceutical composition of the ninth aspect of the invention or an embodiment disclosed herein, for use in treating cancer or a condition involving expression (such as inappropriate expression) of ofCS, such as a condition selected from the group consisting of arthritis, arthropathy, multiple sclerosis, healing after nerve injury, cartilage repair, wound healing, and psoriasis, in a subject in need thereof. Furthermore, this aspect is also relevant for use in targeting delivery of therapeutics to the placenta, for example in the treatment of pre-eclampsia, this latter approach also being applied to the fifteenth aspect of the invention.

[0150] Apart from the fact that cancers have been demonstrated to (over)express ofCS, the same applies to the further pathologies mentioned above, the target (ofCS) is unlikely to be expressed in significant amounts in healthy tissues, so targeting it appears to be a very safe approach.

[0151] As will be evident from the following examples, the antibodies and antibody variants of the invention target a variety of malignancies, and therefore the antibodies and antibody variants and products derived therefrom are highly suitable for use in methods of treating malignancies. Typically, treatment is performed on mammals, particularly humans, but also on domestic animals such as dogs, cats and horses.

[0152] Regarding the cancer to be treated, the cancer cell is derived from a cancer selected from epithelial tumor, non-epithelial tumor (such as blood cancer), and mixed tumor.Epithelial tumor can be either carcinoma or adenocarcinoma, and non-epithelial tumor or mixed tumor can be either liposarcoma, fibrosarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, glioma, neuroblastoma, medulloblastoma, malignant melanoma, malignant meningioma, neurofibrosarcoma, leukemia, myeloproliferative disease, lymphoma (such as B-cell or T-cell lymphoma), angiosarcoma, Kaposi's sarcoma, malignant teratoma, dysgerminoma, seminoma, mesothelioma, or chondrosarcoma melanoma. Also, the anatomical site of the cancer can vary: it can be of the lung, eye, nose, mouth, tongue, pharynx, esophagus, stomach, colon, rectum, bladder, ureters, urethra, kidney, liver, pancreas, thyroid, adrenal glands, breast, skin, central nervous system, peripheral nervous system, meninges, vasculature, testes, prostate, ovaries, uterus, cervix, spleen, bone, or cartilage.

[0153] A fourteenth aspect of the present invention and its embodiments This important aspect relates to a method of treating cancer in a subject in need thereof, or a method of treating a condition involving expression (such as inappropriate expression) of CS, such as a condition selected from the group consisting of arthritis, arthropathy, multiple sclerosis, healing after nerve injury, cartilage repair, wound healing, and psoriasis, or a method of delivering a therapeutic to the placenta, comprising administering to a subject a therapeutically effective amount of an antibody or antibody variant of the first aspect of the invention or any embodiment thereof, a conjugate or fusion protein of the second aspect of the invention or any embodiment thereof, a CAR-T cell of the fourth aspect of the invention or any embodiment thereof, or a pharmaceutical composition of the ninth aspect of the invention or any embodiment thereof.

[0154] Apart from the fact that cancers have been demonstrated to (over)express ofCS, the same applies to the further pathologies mentioned above, the target (ofCS) is unlikely to be expressed in significant amounts in healthy tissues, so targeting it appears to be a very safe approach.

[0155] Regarding the cancer to be treated, the cancer cell is derived from a cancer selected from epithelial tumor, non-epithelial tumor (such as blood cancer), and mixed tumor.Epithelial tumor can be either carcinoma or adenocarcinoma, and non-epithelial tumor or mixed tumor can be either liposarcoma, fibrosarcoma, chondrosarcoma, osteosarcoma, leiomyosarcoma, rhabdomyosarcoma, glioma, neuroblastoma, medulloblastoma, malignant melanoma, malignant meningioma, neurofibrosarcoma, leukemia, myeloproliferative disease, lymphoma (such as B-cell or T-cell lymphoma), angiosarcoma, Kaposi's sarcoma, malignant teratoma, dysgerminoma, seminoma, mesothelioma, or chondrosarcoma melanoma. Also, the anatomical location of the cancer can vary: it can be cancer of the lung, eye, nose, mouth, tongue, pharynx, esophagus, stomach, colon, rectum, bladder, ureters, urethra, kidney, liver, pancreas, thyroid, adrenal glands, breast, skin, central nervous system, peripheral nervous system, meninges, vasculature, testes, prostate, ovaries, uterus, cervix, spleen, bone, or cartilage.

[0156] Various delivery systems are also known and can be used to administer the antibodies and antibody variants or related pharmaceutical compositions or CAR-T cells, such as encapsulation in liposomes, microparticles, and microcapsules.

[0157] Methods of administration provided herein include, but are not limited to, injection, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural, and mucosal (e.g., intranasal and oral routes). In some embodiments, the antibodies, other molecules, or pharmaceutical compositions provided herein are administered intramuscularly, intravenously, subcutaneously, intravenous, intraperitoneally, orally, intramuscularly, subcutaneously, intracavity, transdermally, or transdermally. The compositions can be administered by any convenient route, e.g., injection or bolus injection, absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and can be administered together with other biologically active agents. Administration can be systemic or local. In addition, pulmonary administration, e.g., by use of an inhaler or nebulizer, and in combination with an aerosolizing agent, can also be employed. In some embodiments, administration is localized to the area requiring treatment, e.g., by local injection, infusion, or implant, the implant being composed of a porous, non-porous, or gelatinous material, including membranes or fibers, such as sialastic membranes. In some embodiments, when administering an antibody or antibody variant described herein, care is taken to use materials to which the antibody or antibody variant does not absorb.

[0158] In some embodiments, the antibody or antibody variants provided herein are formulated into liposomes for targeted delivery. Liposomes are vesicles consisting of concentrically arranged phospholipid bilayers that encapsulate an aqueous phase. Liposomes usually contain various types of lipids, phospholipids, and / or surfactants. The components of liposomes are arranged in a bilayer configuration that resembles the lipid arrangement of biological membranes. Liposomes can be useful delivery vehicles due to their biocompatibility, low immunogenicity, and low toxicity. Methods for preparing liposomes are known in the art.

[0159] Also provided herein is a method for treating cancer patients by administering to the patient a unit dose of the antibody or antibody variant, CAR-T cell, or pharmaceutical composition disclosed as part of the present invention. Unit dose refers to a physically discrete unit suitable as a unitary dose for a subject, each unit containing a predetermined amount of active material calculated to produce a desired therapeutic effect in association with a necessary diluent, i.e., carrier or vehicle.

[0160] Administration is performed in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The dosage depends on the subject to be treated, the capacity of the subject's system to utilize the active ingredient, and the degree of therapeutic effect desired. The precise amount of active ingredient required to be administered depends on the practitioner's judgment and is specific to each individual subject. However, suitable dosage ranges for systemic administration are disclosed herein and depend on the route of administration. Suitable regimes for initial and booster administrations are also contemplated, usually an initial administration followed by repeated administrations at intervals of one or more hours by subsequent infusions or other administrations. Exemplary multiple administrations are described above and are useful for maintaining continuously high serum and tissue levels of the polypeptide or antibody. Alternatively, continuous intravenous infusion sufficient to maintain blood concentrations within the ranges specified for the internal therapy is contemplated.

[0161] The therapeutically effective amount is a predetermined amount calculated to achieve the desired effect. Generally, the dosage varies according to the age, condition, sex, and degree of disease of the patient, and can be determined by those skilled in the art. The dosage can be adjusted by individual physicians in the event of complications.

[0162] The exact dosage to be used in the administered formulation also depends on the route of administration and the severity of the disease, and should be determined according to the judgment of the physician and the circumstances of each patient. Effective dosages can be extrapolated from dose-response curves obtained from in vitro or animal model test systems. Dosages are detailed above in the description of the pharmaceutical compositions of the present invention. Usually, the dosage administered to a patient is 0.01 mg / kg to 100 mg / kg of the patient's body weight. In some embodiments, the dosage administered to a patient is 0.01 mg / kg to 20 mg / kg, 0.01 mg / kg to 10 mg / kg, 0.01 mg / kg to 5 mg / kg, 0.01 to 2 mg / kg, 0.01 to 1 mg / kg, 0.01 mg / kg to 0.75 mg / kg, 0.01 mg / kg to 0.5 mg / kg, 0.01 mg / kg to 0.25 mg / kg, 0.01 to 0.15 mg / kg, 0.01 to 0.10 mg / kg, 0.01 to 0.05 mg / kg, or 0.01 to 0.025 mg / kg of the patient's body weight. In particular, the dosage administered to a patient can be 0.2 mg / kg, 0.3 mg / kg, 1 mg / kg, 3 mg / kg, 6 mg / kg, or 10 mg / kg. Even a dose as low as 0.01 mg / kg is expected to show significant pharmacodynamic effects. Dosage levels of 0.10-1 mg / kg are expected to be most appropriate. Higher doses (e.g., 1-30 mg / kg) are also expected to be effective. In general, human and humanized antibodies have a longer half-life in the human body than antibodies of other species due to the immune response to foreign polypeptides. Thus, the dosage of human antibodies can be reduced and the frequency of administration can be reduced. Additionally, the dosage and frequency of administration of the antibodies or antibody variants provided herein can be reduced by enhancing uptake and tissue penetration of the antibodies by modifications such as, for example, lipidation.

[0163] In yet another embodiment, delivery can be performed in a controlled release or sustained release system.Any technique known to those skilled in the art can be used to manufacture sustained release formulations comprising one or more antibodies, molecules, or pharmaceutical compositions provided herein.In one embodiment, a pump can be used in the controlled release system.In another embodiment, a polymeric material can be used to achieve the controlled release of the antibody.

[0164] Examples of polymers that can be used in sustained release formulations include, but are not limited to, poly(hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In yet another embodiment, the controlled release system can be placed close to the treatment target (e.g., lungs), requiring only a fraction of the systemic dose. In another embodiment, a polymer composition useful as a controlled release implant is used according to Dunn et al. (see U.S. Pat. No. 5,945,155). Based on the therapeutic effect of the in situ controlled release of bioactive substances from the polymer system, the implantation can be performed anywhere in the body of a patient requiring therapeutic treatment.

[0165] In another embodiment, a non-polymeric sustained delivery system is used, whereby a non-polymeric implant in the subject's body is used as a drug delivery system. When implanted in the body, the organic solvent of the implant dissipates, disperses, or leaches out of the composition into the surrounding tissue fluid, and the non-polymeric material gradually coagulates or precipitates to form a solid microporous matrix (see U.S. Patent No. 5,888,533). Any technique known to those skilled in the art can be used to manufacture sustained release formulations that include one or more therapeutic agents provided herein.

[0166] The subject treatment includes a single treatment or a series of treatments. It is contemplated that administration can be systemic or local to treat disease, such as inhibiting tumor cell growth or killing cancer cells in cancer patients with locally advanced or metastatic cancer. Administration can be intravenous, intrathecal, and / or intraperitoneal. Furthermore, the antibodies and variants can be administered alone or in combination with anti-proliferative drugs. In this context, any of the fusion and binding partners described above in the context of fusion partners for the antibodies and antibody variants of the invention can be used. In one embodiment, they are administered to reduce the patient's cancer burden before surgery or other procedures. Alternatively, they can be administered after surgery to ensure that any remaining cancer (e.g., cancer that could not be removed by surgery) does not survive. In some embodiments, they can be administered after regression of the primary cancer to prevent metastasis.

[0167] Thus, the antibodies, antibody variants, pharmaceutical compositions and CAR-Ts of the invention can be administered in combination with a second therapy, in some embodiments, the second therapy is an anti-cancer therapy or an anti-hyperproliferative therapy.

[0168] In some embodiments, the compositions and methods comprising administration of the antibodies or antibody variants or CAR-T provided herein, when used in combination with another anti-cancer or anti-hyperproliferative therapy, can enhance the therapeutic efficacy of the other anti-cancer or anti-hyperproliferative therapy. Thus, the methods and compositions described herein can be provided in combination with a second therapy to achieve a desired effect, such as killing cancer cells, inhibiting cell hyperproliferation, and / or inhibiting cancer metastasis.

[0169] In some embodiments, the second therapy has a direct cytotoxic effect, such as chemotherapy, targeted therapy, cryotherapy, hyperthermia, photodynamic therapy, high intensity focused ultrasound (HIFU) therapy, radiation therapy, or surgical therapy. Targeted therapy can be biological targeted therapy or small molecule targeted therapy. In other embodiments, the second therapy does not have a direct cytotoxic effect. For example, the second therapy can be a drug that upregulates the immune system without having a direct cytotoxic effect.

[0170] Provided herein are methods that include administering the antibodies, antibody variants, pharmaceutical compositions and CAR-T of the present invention in combination with a second or additional therapy. Administration can occur before, during, after, or in various combinations relative to the second anti-cancer therapy. Administration can occur simultaneously to intervals ranging from minutes to days to weeks. In some embodiments in which the antibodies, antibody variants, pharmaceutical compositions and CAR-T described herein are provided to a patient separately from the second anti-cancer agent, typically no significant period of time will elapse between the time of each administration, allowing the two compounds to still exert their beneficial combined effect on the patient. In such cases, it is contemplated that the antibodies, antibody variants, pharmaceutical compositions and CAR-T described herein, and the second anti-cancer therapy, can be provided to the patient within about 12-24 or 72 hours of each other, more specifically within about 6-12 hours of each other. In some circumstances, the duration of treatment can be significantly extended, with days (2, 3, 4, 5, 6, or 7) to weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapse between each administration.

[0171] In certain embodiments, the treatment course lasts for 1-90 days or more (including intervening days). It is contemplated that one agent is administered on any day from day 1 to day 90 (including intervening days) or any combination thereof, and another agent is administered on any day from day 1 to day 90 (including intervening days) or any combination thereof. Within a day (24 hours), the patient is administered one or more doses of the agent. It is further contemplated that the treatment course is followed by a period during which no anti-cancer treatment is administered. This period may last for 1-7 days, and / or 1-5 weeks, and / or 1-12 months or more (including intervening days), depending on the patient's prognosis, strength, health, and other conditions. The treatment cycle can be repeated as necessary.

[0172] Fifteenth aspect of the present invention and its embodiments This aspect is a method for detecting ofCS in a sample, the method comprising: 1) contacting said sample with an antibody or antibody variant of the first aspect of the invention or any aspect thereof disclosed herein, to allow the antibody or antibody variant to specifically bind to ofCS, and subsequently contacting the antibody or antibody variant and ofCS bound thereby with an agent capable of binding to ofCS, to allow the agent to bind to ofCS; or 2) contacting said sample with an agent capable of binding to ofCSPG, allowing the agent to bind to ofCS, and subsequently contacting the agent and the ofCS bound thereby with an antibody or antibody variant of the first aspect of the invention or any aspect thereof disclosed herein, allowing the antibody to bind to ofCS; and quantitatively or qualitatively detecting the presence of a complex comprising the ofCS, the antibody or antibody variant, and the drug. wherein the agent and the antibody or antibody variant are competitive or non-competitive for binding to ofCS.

[0173] One very attractive feature of the antibodies identified as part of this invention is that they do not all compete for binding to the same epitope on the ofCS. This allows antibodies to be used in a set-up where one acts as the capture antibody (usually bound to a solid or semi-solid surface) and the other acts as the detection / detectable antibody, without the antibodies displacing each other and causing inaccuracies in the assay. This facilitates direct assays; other antibodies compete for binding to the ofCS, allowing for competitive assay formats.

[0174] The sample is typically a sample comprising or consisting of cells and / or tissue, preferably body fluids such as blood, urine, saliva, central nervous system fluids, lymph, feces, and biopsies. The sample is typically diluted in a suitable sampling liquid, such as a sampling buffer.

[0175] The agent of option 1 is, in one embodiment, an antibody or antibody fragment of the first aspect of the invention and any embodiment thereof, VAR2CSA (SEQ ID NO: 135) or a CS-binding fragment thereof (such as a protein consisting of or including SEQ ID NO: 136), an antibody that binds to chondroitin sulfate, an antibody that binds to the protein core of a proteoglycan (a particularly preferred possibility), or a glycosaminoglycan staining dye.

[0176] In option 2, the agent is typically VAR2CSA (SEQ ID NO: 135) or a CSA-binding fragment thereof (such as SEQ ID NO: 136), or an antibody that binds to the protein core of the proteoglycan.

[0177] As mentioned above, the agent or antibody or antibody variant is conveniently bound to a solid or semi-solid phase.

[0178] Detection of the complex is also preferably facilitated by labeling the antibody or agent with a detectable moiety or by adding a detectable agent that binds to the antibody or agent, however, it is also possible to detect the complex by mass spectrometry methods well known in the art.

[0179] Conventional assay formats can be used: enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), real-time immunoquantitative PCR (iqPCR), microarrays using fluorescent reporters, electrochemiluminescence tag assays, and plasmon resonance formats are all useful.

[0180] The latter (plasmon resonance) is a very simple assay format that only requires the binding of the antibody / antibody variant to the target and therefore only requires the presence of the antibody or variant of the invention as a capture agent for ofCS. Thus, essentially, the assay is limited to contacting the antibody or antibody variant of the invention with the sample and then measuring the interaction via plasmon resonance technology.

[0181] Sixteenth aspect of the present invention and its embodiments This aspect relates to a detection kit comprising an antibody of the first aspect of the invention or any embodiment thereof disclosed herein, an agent as defined in the fourteenth aspect of the invention or any embodiment thereof disclosed herein, and a reaction vessel, and optionally a labelling agent which binds to the antibody or the agent.

[0182] Embodiments of this aspect include any conventional assay components, i.e., the reaction vessel can be an ELISA plate, a hollow fiber device, a lateral flow device, a dipstick, a microarray, and such assay vessels are known to those of skill in the art.

[0183] Further components of such kits may be various detection means (fluorescent or luminescent probes, radioactive labels, staining agents, etc.) as well as reaction buffers, washing solutions, and the like.

[0184] Seventeenth aspect of the invention and its embodiments This aspect relates to a method of providing a nucleic acid encoding an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, comprising screening a library comprising antigen-binding fragments of the antibody, the library being comprised of a display agent selected from cells, viruses and phages comprising nucleic acid fragments encoding the antibody, such that the display agent binds to a capture agent, the capture agent consisting essentially of purified ofCS or ofCS-decorated protein, subsequently isolating the nucleic acid fragments encoding the antibody from the capture agent-bound display agent and optionally sequencing the isolated nucleic acid fragments.

[0185] Preferred display agents are selected from yeast cells (when utilizing yeast display technology for antibody identification) or phages selected from phages M3, fd, T4, T7 and lambda (when utilizing phage display technology for antibody identification).

[0186] In either case, the antigen-binding fragment of the antibody is typically in the format of scFV, F(ab), F(ab')2, and F(ab'). In some embodiments, the antigen-binding fragment comprises synthetic, semi-synthetic, or CDRs derived from a human antibody library.

[0187] In relation to the seventeenth aspect, based on the recognition that ofCS-specific antibodies can be identified and isolated via phage display, the inventors have also recognized that aptamer libraries can be screened for binders of ofCS in essentially the same manner as phage display libraries are screened. Thus, such methods for the identification and isolation of ofCS-binding aptamers are also part of the present invention, as are aptamers that share the binding properties of the antibodies of the present invention.

[0188] The eighteenth aspect of the present invention and its embodiments This aspect relates to a method of producing an antibody or antibody variant of the first aspect of the invention or any embodiment thereof disclosed herein, comprising expressing in a host cell in culture an expression vector comprising a nucleic acid fragment obtained according to the method of the first aspect of the invention or any embodiment thereof disclosed herein, and subsequently isolating the expression product from the culture.

[0189] The method of the seventeenth aspect will typically provide as main output the identification of the variable domains of the antibody, which will enable the skilled person to incorporate the variable domains (or, where appropriate, CDRs) into the framework of a desired format such as IgG or other antibody / antibody variant formats as described herein (which may be obtained by recombinant expression of the coding sequences in a host cell).

[0190] Biological sequences The amino acid sequences referred to in the specification and claims are as follows: F1 (SEQ ID NO:1) DIVMTQSPDSLAVSLGERATINCKSSQSVLGSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYRSPYTFGQGTKVEIKGGGGGSGG GGSGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAPGKGLEWVSRISGSGGQTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGHSSSWLRGH FQHWGQGTLVTVSS F1-CDR-L1 (SEQ ID NO:2) KSSQSVLGSSNNKNYLA F1-CDR-L2 (SEQ ID NO:3) WASTRES F1-CDR-L3 (SEQ ID NO: 4) QQYYRSPYT F1-CDR-H1 (SEQ ID NO:5) GFTFSDYAMS F1-CDR-H2 (SEQ ID NO:6) RISGSGGQTKYADSVKG F1-CDR-H3 (SEQ ID NO: 7) GHSSSWLRGHFQH F2 (SEQ ID NO:8) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYDASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQQYNYPYTFGQGTKVEIKGGGGGSGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTSSAMSWVRQAPGKGLEWVSRIDGSGGDTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGRRLWFGDPFDHWGQGTLVTVSS F2-CDR-L1 (SEQ ID NO: 9) KSSQSVLRSGNNKNRLA F2-CDR-L2 (SEQ ID NO: 10) DASTRES F2-CDR-L3 (SEQ ID NO: 11) QQQYNYPYT F2-CDR-H1 (SEQ ID NO: 12) GFTFTSSAMS F2-CDR-H2 (SEQ ID NO: 13) RIDGSGGDTRYADSVKG F2-CDR-H3 (SEQ ID NO: 14) GRRLWFGDPFDH F3 (SEQ ID NO: 15) DIVMTQSPDSLAVSLGERATINCKSSQSVLSSSSNNKNRLAWYQQKPGQPPKLLIYAASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYSSPWTFGQGTKVEIKGGGGSGGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYGMSWVRQAPGKGLEWVSRISGSGGYTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVYFDGTTRYFDSWGPGTLVTVSS F3-CDR-L1 (SEQ ID NO: 16) KSSQSVLSSSNNKNRLA F3-CDR-L2 (SEQ ID NO: 17) AASTRES F3-CDR-L3 (SEQ ID NO: 18) QQGYSSPWT F3-CDR-H1 (SEQ ID NO: 19) GFTFSNYGMS F3-CDR-H2 (SEQ ID NO: 20) RISGSGGYTNYADSVKG F3-CDR-H3 (SEQ ID NO:21) VYFDGTTRYFDS F4 (SEQ ID NO:22) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYAASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYRAPITFGQGTKVEIKGGGGSGGGGSGGGSGG GGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYAMSWVRQAPGKGLEWVSAIDGGGDSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAKFGSRRPERGMGYFDLWGRGTLVTVSS F4-CDR-L1 (SEQ ID NO:23) KSSQSVLRSGNNKNRLA F4-CDR-L2 (SEQ ID NO:24) AASTRES F4-CDR-L3 (SEQ ID NO:25) QQSYRAPIT F4-CDR-H1 (SEQ ID NO:26) GFTFTDYAMS F4-CDR-H2 (SEQ ID NO:27) AIDGGGDSTYYADSVKG F4-CDR-H3 (SEQ ID NO:28) AKFGSRRPERGMGYFDL F5 (SEQ ID NO:29) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSSNNKNRLAWYQQKPGQPPKLLIYAASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYRAPFTFGQGTKVEIKGGGGGSGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSRIDGSGGTTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRGYNYGYFDYWGQGTLVTVSS F5-CDR-L1 (SEQ ID NO: 30) KSSQSVLRSSNNKNRLA F5-CDR-L2 (SEQ ID NO:31) AASTRES F5-CDR-L3 (SEQ ID NO:32) QQGYRAPFT F5-CDR-H1 (SEQ ID NO: 33) GFTFSNYAMS F5-CDR-H2 (SEQ ID NO:34) RIDGSGGTTNYADSVKG F5-CDR-H3 (SEQ ID NO: 35) SRGYNYGYFDY F6 (SEQ ID NO:36) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSSNNKNRLAWYQQKPGQPPKLLIYAASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYNAPLTFGQGTKVEIKGGGGSGGGGSGG GSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTNYAMSWVRQAPGKGLEWVSRISGSGSSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARFRRLRSLYFDYWGQGTLVTVSS F6-CDR-L1 (SEQ ID NO:37) KSSQSVLRSSNNKNRLA F6-CDR-L2 (SEQ ID NO:38) AASTRES F6-CDR-L3 (SEQ ID NO: 39) QQGYNAPLT F6-CDR-H1 (SEQ ID NO: 40) GFTFTNYAMS F6-CDR-H2 (SEQ ID NO: 41) RISGSGSSTSYADSVKG F6-CDR-H3 (SEQ ID NO: 42) FRLRSLYFDY F7 (SEQ ID NO:43) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSSNNKNRLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYNSPLTFGQGTKVEIKGGGGSGGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTNYAMSWVRQAPGKGLEWVSWISGRGGSTNYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARARARRSSGYFDYWGQGTLVTVSS F7-CDR-L1 (SEQ ID NO: 44) KSSQSVLRSSNNKNRLA F7-CDR-L2 (SEQ ID NO: 45) GASTRES F7-CDR-L3 (SEQ ID NO: 46) QQGYNSPLT F7-CDR-H1 (SEQ ID NO: 47) GFTFTNYAMS F7-CDR-H2 (SEQ ID NO: 48) WISGRGGSTNYADSVKG F7-CDR-H3 (SEQ ID NO: 49) ARARSSGYFDY F8 (SEQ ID NO:50) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYSSPITFGQGTKVEIKGGGGSE VQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSRIDGGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVRYPTGPYYFDYWGQGTLVTVSS F8_18AA linker (SEQ ID NO:51) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYSSPITFGQGTKVEIKGGGGSGGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSDYYMSWVRQAPGKGLEWVSRIDGGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVRYPTGPYYFDYWGQGTLVTVSS F8-CDR-L1 (SEQ ID NO:52) KSSQSVLRSGNNKNRLA F8-CDR-L2 (SEQ ID NO:53) GASTRES F8-CDR-L3 (SEQ ID NO:54) QQGYSSPIT F8-CDR-H1 (SEQ ID NO:55) GFTFSDYYMS F8-CDR-H2 (SEQ ID NO:56) RIDGGGGSTYYADSVKG F8-CDR-H3 (SEQ ID NO:57) VRYPTGPYYFDY F9 (SEQ ID NO:58) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSNNNKNRLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYSYPLTFGQGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLFESGGGLVQPGGSLRLSCAASGFTFTDYAMSWVRQAPGKGLEWVSAIQGGGGGTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRRPTVTTSYFDYWGQGTLVTVSS F9-CDR-L1 (SEQ ID NO:59) KSSQSVLRSNNNKNRLA F9-CDR-L2 (SEQ ID NO:60) GASTRES F9-CDR-L3 (SEQ ID NO:61) QQGYSYPLT F9-CDR-H1 (SEQ ID NO:62) GFTFTDYAMS F9-CDR-H2 (SEQ ID NO:63) AIQGGGGGTRYADSVKG F9-CDR-H3 (SEQ ID NO:64) DRRPTVTTSYFDY F10 (SEQ ID NO:65) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSYNRPLTFGQGTKVEIKGGGGGSGGGGSG GGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYQMSWVRQAPGKGLEWVSEISGYGGSTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRNAAFDIWGQGTMVTVSS F10-CDR-L1 (SEQ ID NO:66) KSSQSVLRSGNNKNRLA F10-CDR-L2 (SEQ ID NO:67) WASTRES F10-CDR-L3 (SEQ ID NO:68) QQSYNRPLT F10-CDR-H1 (SEQ ID NO:69) GFTFSSYQMS F10-CDR-H2 (SEQ ID NO: 70) EIS GYGGSTKYADSVKG F10-CDR-H3 (SEQ ID NO:71) RNAAFDI F11 (SEQ ID NO:72) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSDNNENRLAWYQQKPGQPPKLLIYAASTRESGVPDRFSGSGSGTFTLTISSLQAEDVAVYYCQQYYREPITFGQGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTSYGMSWVRQAPGKGLEWVSGISGRGGSTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTRGRIAARRGWFDPWGQGTLVTVSS F11-CDR-L1 (SEQ ID NO: 73) KSSQSVLRSDNNENRLA F11-CDR-L2 (SEQ ID NO:74) AASTRES F11-CDR-L3 (SEQ ID NO: 75) QQYYREPIT F11-CDR-H1 (SEQ ID NO:76) GFTFTSYGMS F11-CDR-H2 (SEQ ID NO:77) GISGRGGSTSYADSVKG F11-CDR-H3 (SEQ ID NO:78) TRGRIAARRGWFDP F12 (SEQ ID NO:79) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSDNNKNRLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYSSPWTFGQGTKVEIKGGGGSGGGGSGGSG GGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTSTAMSWVRQAPGKGLEWVSAISGRGGSTKYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARASKKSPIAAAGTFDLWGQGTLVTVSS F12-CDR-L1 (SEQ ID NO: 80) KSSQSVLRSDNNKNRLA F12-CDR-L2 (SEQ ID NO: 81) GASTRES F12-CDR-L3 (SEQ ID NO:82) QQGYSSPWT F12-CDR-H1 (SEQ ID NO: 83) GFTFTSTAMS F12-CDR-H2 (SEQ ID NO: 84) AISGRGGSTKYADSVKG F12-CDR-H3 (SEQ ID NO: 85) ASKKSPIAAAGTFDL F13 (SEQ ID NO:86) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNRPYTFGQGTKVEIKGGGGGSGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTDYGMSWVRQAPGKGLEWVSGISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSRRRDYNWFDPWGQGTLVTVSS F13-CDR-L1 (SEQ ID NO:87) KSSQSVLRSGNNKNRLA F13-CDR-L2 (SEQ ID NO:88) GASTRES F13-CDR-L3 (SEQ ID NO:89) QQYYNRPYT F13-CDR-H1 (SEQ ID NO: 90) GFTFTDYGMS F13-CDR-H2 (SEQ ID NO: 91) GISGSGGSTYYADSVKG F13-CDR-H3 (SEQ ID NO: 92) SRRRDYNWFDP F14 (SEQ ID NO:93) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYAASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYREPLTFGQGTKVEIKGGGGSGGGGSGGGS GGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVSAISGSGGQTSYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHRRGRGAMGYFDYWGQGTLVTVSS F14-CDR-L1 (SEQ ID NO:94) KSSQSVLRSGNNKNRLA F14-CDR-L2 (SEQ ID NO: 95) AASTRES F14-CDR-L3 (SEQ ID NO: 96) QQGYREPLT F14-CDR-H1 (SEQ ID NO: 97) GFTFSNYYMS F14-CDR-H2 (SEQ ID NO: 98) AISGSGGQTSYADSVKG F14-CDR-H3 (SEQ ID NO: 99) HRRGRGAMGYFDY F15 (SEQ ID NO: 100) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYAASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYSYPYTFGQGTKVEIKGGGGGSGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSDIDGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDRRARSHYFDYWGQGTLVTVSS F15-CDR-L1 (SEQ ID NO: 101) KSSQSVLRSGNNKNRLA F15-CDR-L2 (SEQ ID NO: 102) AASTRES F15-CDR-L3 (SEQ ID NO: 103) QQGYSYPYT F15-CDR-H1 (SEQ ID NO: 104) GFTFSSYGMS F15-CDR-H2 (SEQ ID NO: 105) DIDGSGGSTYYADSVKG F15-CDR-H3 (SEQ ID NO: 106) DRRARSHYFDY F16 (SEQ ID NO:107) DIVMTQSPDSLAVSLGERATINCKSSQSVLSSYNNKNRLAWYQQKPGQPPKLLIYAASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYSRPITFGQGTKVEIKGGGGGSGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFTSYQMSWVRQAPGKGLEWVSVIDGSGGYTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASPRRGKGSYFDYWGQGTLVTVSS F16-CDR-L1 (SEQ ID NO: 108) KSSQSVLSSYNNKNRLA F16-CDR-L2 (SEQ ID NO: 109) AASTRES F16-CDR-L3 (SEQ ID NO: 110) QQGYSRPIT F16-CDR-H1 (SEQ ID NO: 111) GFTFTSYQMS F16-CDR-H2 (SEQ ID NO: 112) VIDGSGGYTRYADSVKG F16-CDR-H3 (SEQ ID NO: 113) PRRGKGSYFDY F17 (SEQ ID NO:114) DIVMTQSPDSLAVSLGERATINCKSSQSVLRSGNNKNRLAWYQQKPGQPPKLLIYGASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQGYSAPITFGQGTKVEIKGGGGSGGGGGSGGG SGGGGSEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTRYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARARSSSSGGYFDYWGQGTLVTVSS F17-CDR-L1 (SEQ ID NO: 115) KSSQSVLRSGNNKNRLA F17-CDR-L2 (SEQ ID NO: 116) GASTRES F17-CDR-L3 (SEQ ID NO: 117) QQGYSAPIT F17-CDR-H1 (SEQ ID NO: 118) GFTFSSYAMS F17-CDR-H2 (SEQ ID NO: 119) AISGSGGSTRYADSVKG F17-CDR-H3 (SEQ ID NO: 120) ARSSSGGYFDY C9-VLVH (SEQ ID NO:121) QPVLTQPPSASGTPGQRVTISSCSGSSSNIGNNPVNWYQQLPGTAPKLLIYRNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEAHYYCAAWNDRLSGPRVFGGGTKLTVLGGGGSGGGGSG GGGSGGGQVQLVQSGGGLAQPGGSLRLSCAASGFTFRSYWMTWARQAPGKGLEWVATIKQDGSEKYYVDSVKGRFTISRDDATNSLFLQMNSLRAEDTALYYCARARNMDVWGQGTTVTVSS C9-CDR-L1 (SEQ ID NO: 122) SGSSSNIGNNPVN C9-CDR-L2 (SEQ ID NO: 123) RNNQRPS C9-CDR-L3 (SEQ ID NO: 124) AAWNDRLSGPRV C9-CDR-H1 (SEQ ID NO: 125) GFTFRSYWMT C9-CDR-H2 (SEQ ID NO: 126) TIKQDGSEKYYVDSVKG C9-CDR-H3 (SEQ ID NO: 127) ARNMDV B3-VLVH (SEQ ID NO:128) DIVMTQSPSSMSASVGDRVTITCRASQGLSNRLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTFTLTISRLQPEDFATYYCQQANSFPLTFGQGTRLEIKGGGGSGGGGSGGGSGG GGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARGGSSGWRKYFQHWGQGTLVTVSS B3-CDR-L1 (SEQ ID NO: 129) RASQGLSNRLA B3-CDR-L2 (SEQ ID NO: 130) AASSLQS B3-CDR-L3 (SEQ ID NO: 131) QQANSFPLT B3-CDR-H1 (SEQ ID NO: 132) GYTFTGYYMH B3-CDR-H2 (SEQ ID NO: 133) WINPNSGGTNYAQKFQG B3-CDR-H3 (SEQ ID NO: 134) GGSSGWRKYFQH VAR2CSA (SEQ ID NO:135) VAR2CSA-ID1-ID2a (SEQ ID NO: 136) NYIKGDPYFAEYATKLSFILNPSDANNPSGETANHNDEACNCNCNESGISSVGQAQTSGPSSNKTCITHSSIKTNKKKECKDVKLGVRENDKDLKICVIEDTSLSGVDNCCCQDLLGILQENCSDNKRGSSSNDSCDNKNQDECQKKLEKVFASLTNGYKCD KCKSGTSRSKKKWIWKKSSGNEEGLQEEYANTIGLPPRTQSLYLGNLPKLENVCEDVKDINFDTKEKFLAGCLIVSFHEGKNLKKRYPQNKNSGNKENLCKALEYSFADYGDLIKGTSIWDNEYTKDLELNLQNNFGKLFGKYIKKNNTAEQDTSYSSLD ELRESWWNTNKKYIWTAMKHGAEMNITTCNADGSVTGSGSSCDDIPTIDLIPQYLRFLQEWVENFCEQRQAKVKDVITNCKSCKESGNKCKTECKTKCKDECEKYKKFIEACGTAGGGIGTAGSPWSKRWDQIYKRYSKHIEDAKRNRKAGTKNCGTSST TNAAASTDENKCVQSDIDSFFKHLIDIGLTTPSSYLSNVLDDNICGADKAPWTTYTTYTTTEKCNKERDKSKSQSSDTLVVVNVPSPLGNTPYRYKYACQCKIPTNEETCDDRKEYMNQWSCGSARTMKRGYKNDNYELCKYNGVDVKPTTVRSNSSKLD GS-Linker-1 (SEQ ID NO:137) GGGGSGGGGSGGGSGGGGS GS-Linker-2 (SEQ ID NO:138) GGGGSGGGGSGGGGSGGG GS-Linker-3 (SEQ ID NO:139) GGGSGGGS

[0191] Generally, when referring to the antibodies or antibody variants of the invention by name (i.e., F1-F17, C9, and B3), a general, non-specific reference to such an antibody name (such as "C9" or the "C9 antibody") is intended to relate generally to any antibody format or antibody variant that contains the six corresponding CDR sequences described above. Thus, unless more specific details regarding a particular construct format are given, it is as follows:

[0192] "F1 antibody" or "F1" refers to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NO:1 as well as SEQ ID NO:2-7. "F2 antibody" or "F2" refers to the antibody or antibody variant format described herein that contains the six CDR sequences set forth in SEQ ID NO: 9-14 as well as SEQ ID NO: 8. "F3 antibody" or "F3" refers to the antibody or antibody variant format described herein that contains not only SEQ ID NO:15, but also the six CDR sequences set forth in SEQ ID NOs:16-21. "F4 antibody" or "F4" refers to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NO:22 as well as SEQ ID NO:23-28. "F5 antibody" or "F5" refers not only to the polypeptide consisting of SEQ ID NO:29, but also to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NOs:30-35. "F6 antibody" or "F6" refers not only to the polypeptide consisting of SEQ ID NO:36, but also to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NOs:37-42. "F7 antibody" or "F7" refers not only to the polypeptide consisting of SEQ ID NO:43, but also to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NOs:44-49. "F8 antibody" or "F8" refers not only to the polypeptide consisting of SEQ ID NO:50, but also to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NOs:52-57. "F9 antibody" or "F9" refers not only to the polypeptide consisting of SEQ ID NO:58, but also to any antibody or antibody variant format described herein that contains the six CDR sequences set forth in SEQ ID NOs:59-64. "F10 antibody" or "F10" refers not only to the polypeptide consisting of SEQ ID NO:65, but also to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NOs:66-71. "F11 antibody" or "F11" refers not only to the polypeptide consisting of SEQ ID NO:72, but also to the antibody or antibody variant format described herein that contains the six CDR sequences set forth in SEQ ID NOs:73-78. "F12 antibody" or "F12" refers not only to the polypeptide consisting of SEQ ID NO:79, but also to the antibody or antibody variant formats described herein that contain the six CDR sequences set forth in SEQ ID NOs:80-85. "F13 antibody" or "F13" refers not only to the polypeptide consisting of SEQ ID NO:86, but also to the antibody or antibody variant formats described herein that contain the six CDR sequences set forth in SEQ ID NOs:87-92. "F14 antibody" or "F14" refers not only to the polypeptide consisting of SEQ ID NO:83, but also to the antibody or antibody variant formats described herein that contain the six CDR sequences set forth in SEQ ID NOs:84-99. "F15 antibody" or "F15" refers not only to the polypeptide consisting of SEQ ID NO:100, but also to the antibody or antibody variant formats described herein that contain the six CDR sequences set forth in SEQ ID NOs:101-106. "F16 antibody" or "F16" refers not only to the polypeptide consisting of SEQ ID NO:107, but also to the antibody or antibody variant formats described herein that contain the six CDR sequences set forth in SEQ ID NOs:108-113. "F17 antibody" or "F17" refers not only to the polypeptide consisting of SEQ ID NO:114, but also to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NOs:115-120. "C9 antibody" or "C9" refers not only to the polypeptide consisting of SEQ ID NO:121, but also to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NOs:122-127. "B3 antibody" or "B3" refers not only to the polypeptide consisting of SEQ ID NO:128, but also to the antibody or antibody variant forms described herein that contain the six CDR sequences set forth in SEQ ID NOs:129-134. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS EXAMPLES

[0193] Identification ofCS-specific antibodies The obvious starting point for generating high-affinity IgG monoclonal antibodies against ofCS was to immunize mice with purified ofCS using an immunization strategy capable of breaking mouse self-tolerance, followed by the use of ex vivo hybridoma or EBV transformation techniques to generate monoclonal antibodies. However, multiple attempts using mouse immunization against biotinylated placental CS or recombinant ofCS-carrying proteoglycans (on decorin or serglycin scaffolds) failed to identify high-affinity, highly specific ofCS-specific antibodies. Mouse sera obtained after a series of immunizations were tested using both antigen ELISA and flow cytometry against ofCS-positive cancer cells, but no clones were identified that bound significantly relative to controls. Further screening of monoclonals using FACS sorting did not find any specific clones. Multiple iterations of adjuvant strain modification paralleled by modification of the binding strategy of ofCS to keyhole limpet hemocyanin, a highly immunogenic T-cell-dependent antigen, did not yield ofCS-specific monoclonal antibodies. Furthermore, attempts at binding to virus-like particles did not yield significantly positive immune sera. Repeated immunizations in rats and chickens to generate ofCS-specific IgY were unsuccessful.

[0194] A breakthrough was only achieved when the strategy was modified to use phage display. The key to success in generating ofCS-specific antibodies using phage display was the combination of a panning / selection strategy using precise ofCS epitopes available for panning and the proven rVAR2 specificity for ofCS. The key reagents for the panning procedure were highly purified and well-characterized ofCS and recombinant ofCSPGs that uniformly display the ofCS modifications on the decorin backbone. To this end, the decorin gene was cloned into the pCDNA3.1 vector under a CMV promoter with a 6XHis tag and produced as a secreted form in Chinese hamster ovary cells. The protein was purified using IMAC chromatography and desalted in PBS. The protein was validated using SDSPAGE and binding to VAR2CSA protein with or without chondroitinase ABC (ChABC) treatment. Binding of VAR2CSA to recombinant decorin was relatively lower than that to native decorin; the reason was identified to the low levels of ofCS using both SDSPAGE and Alcian blue staining. This could be an artifact from recombinant expression, where the ofCS enzymatic machinery cannot keep up with the gene expression of the protein backbone. The challenge of low abundance of CS on recombinantly produced proteins was resolved by enriching for CS. CS-enriched decorin was obtained using anion exchange chromatography, taking advantage of the high negative charge of CS. OfCS from placental tissue was purified and quality controlled as described by eaudet et al., Glycoconj. J., 2014. Biotinylated ofCS or recombinant ofCSPGs were used for biopanning of phage display libraries constructed from the complete naive Homosapiens immunoglobulin repertoire, fully synthetic Fabs, or semi-synthetic ScFvs with combinatorial variations of over 1012 clones. Negative selection was performed in an iterative process using chondroitinase ABC treatment of the reagents before negative selection and / or purified heparan sulfate proteoglycans (HSPGs) as a source of highly charged non-ofCS glycans.Elution of bound phages was performed by pH elution or by competition with rVAR2 competing for the ofCS epitope. After the third and fourth rounds of biopanning, selected clones were tested for binding to ofCS in ELISA. Specificity was also verified by testing binding to different sources of ofCS, HSPGs, and by competitive ELISA with rVAR2. Deep sequencing of selected phages was performed to reliably identify all clones. In total, 19 antibody fragments with specificity for ofCS when presented in a phage context were obtained from six different phage display libraries. EXAMPLES

[0195] Production of antibodies and antibody fragments scFv expression and purification: The amino acid sequences of the ofCS-binding VL and VH domains derived from phage display were generated as single-chain variable fragments (scFv) in E. coli cells. The VL and VH domains were genetically fused using an 18 amino acid linker. A V5 tag, a 6xHIS tag and a SpyTag were added to the C-terminus of the scFv. The scFvs were expressed in E. coli Shuffle cells (NEB) and purified from the soluble fraction after cell lysis. A two-step purification was performed using cation exchange chromatography using a HisTrap HP (Cytiva) column for capture followed by a HiTrap SP HP column (Cytiva) for polishing.

[0196] Dimeric SpyCatcher expression and purification: SpyCatcher molecules were cloned and expressed as dimeric constructs. A short flexible linker, GGGSGGGS (SEQ ID NO: 139), was used to join two SpyCatcher molecules. A 6xHIS tag was also added to the C-terminus. The constructs were expressed in E. coli BL21 cells and purified from the soluble fraction after cell lysis. A two-step purification was performed using a HisTrapHP (Cytiva) column for capture, followed by anion exchange chromatography using a HiTrapQHP column (Cytiva) for polishing.

[0197] IgG expression and purification: Fully human IgG antibodies containing ofCS VL and VH domains for binding were produced in CHO cells. Target VH and VL domains were ordered as separate genes and subcloned into pTRIOZ (Invivogen) vectors containing separate cassettes for the heavy and light chains for human IgG1 kappa expression. Protein expression was achieved using the expiCHO transient transfection system (ThermoFisher) following the product guidelines. Media was harvested 7-9 days after transfection and the produced IgG was captured using a Protein A column. The protein was further purified using an anion exchange column in flow-through mode to obtain monomeric mAb.

[0198] Anti-ofCSscFv was produced in high yields (>20mg / L) and displayed as clean monomeric protein on SDS-page. To promote dimerization, a split protein system based on the spytag sequence was used, which forms an isopeptide bond with the corresponding spycatchertag. Spytagged ofCSscFv was conjugated with dimeric Spycatcher at a 2:1 molar ratio to create a bivalent structure resembling the variable region of IgG. All structures remained soluble and retained ligand binding. Increasing valency from monomer to dimer increased binding. EXAMPLES

[0199] Specificity ofCS antibodies and antibody fragments The specificity of ofCS binding may change when moving from a format in which sequences are displayed at high density on phage to therapeutically relevant molecules such as scFv2, Fab, and IgG. To address this, we tested binding to ofCS by ELISA for both scFv dimers and IgG as described (Salanti and Clausen et al., Cancer Cell, 2015). To determine whether the interaction was indeed between the antibody reagent and the glycosaminoglycan chains, we also tested binding after chondroitinase treatment of ofCS. Figure 1 shows the binding of selected IgGs and Figure 2 shows the binding of selected ScFvs. As a control, rVAR2 (VAR2CSA) was included. Binding to ofCS was confirmed for all antibodies and antibody fragments tested, some to a higher extent than VAR2CSA. Chondroitinase treatment of the target reagent completely inhibited binding, indicating complete specificity for chondroitin-type glycosaminoglycans (Figures 1 and 2). Inhibition using soluble chondroitin sulfate showed mixed and overlapping specificities for CSA, CSC, and CSB (dermatan sulfate) (data not shown), likely indicating the hybrid nature of ofCS described above.

[0200] The antibodies or antibody fragments were then tested for binding to recombinant ofCSPGs (ofCS on a decorin backbone) (Tables 1 and 3) and to commercially available HSPGs from Merck (Sigma-Aldrich; H4777) (Tables 2 and 4). The tables demonstrate limited binding to HSPGs, but a very clear preference for binding to CSPGs. Overall, the data show that the ofCS biopanned antibody fragments and antibodies generated bind specifically to chondroitin sulfate as well as recombinant malaria VAR2 protein, with limited binding to charged HSPG molecules.

[0201] In a competitive ELISA setup, we demonstrated that VAR2CSA and ofCS-binding antibodies share a common epitope. Briefly, ELISA plates were coated with ofCS and binding of ofCS antibodies was measured in the presence or absence of rVAR2. This was made possible by using VAR2 without a V5 tag and anti-ofCSscFv with a V5 tag. As an example, the OD value of F2 anti-ofCS against a 400 nM concentration of ofCS was measured to be 2.0, and the addition of 3 μM VAR2 protein reduced the binding to 1.2 OD. Similarly, the OD value of C9 binding to ofCSPG at 25 nM was 1.4, and the OD reduced to 0.2 upon competition with 3 μM VAR2.

[0202] [Table 1] [Table 2] [Table 3] [Table 4]

[0203] We then obtained all available monoclonal antibodies previously described to bind chondroitin sulfate and tested their binding to ofCS, recombinant ofCSPGs (ofCS on the decorin backbone), and purified decorin from Merck (Sigma-Aldrich). We tested PG4, CS56, 2H6, and BE-123. The latter was treated with chondroitinase, since it only recognizes the digested CS fragment. For comparison, we included the ofCS-binding B3 clone. Very clearly, none of the previously described antibodies bound to ofCS or CSPGs, but to CS presented on purified decorin (except for PG4, which did not bind to any of the coated CS reagents) (Figure 3). This indicates that our panel of antibodies binding to ofCS and recombinant CSPGs is unique compared to published antibodies. Moreover, all of these antibodies were of the IgM type (BE-123 is an IgG, so enzymatic predigestion was necessary).

[0204] After defining the oncofetal specificity of the antibodies, we then determined preferences for distinct sulfation in parallel with oncofetal CS chain modifications. The table below shows the detailed specificity of this panel of preferred antibody fragments, obtained from binding inhibition assays or disaccharide analysis after pull-down. [Table 5]

[0205] Apparently, F3 and F8 have a preference for 6-sulfated (CSC) whereas C9 has mixed specificity, and B3, F11 and others appear to bind preferentially to dermatan sulfate containing GAGs. EXAMPLES

[0206] Determining the affinity of ofCS antibodies to ofCS using biosensors The Attana biosensor system has previously proven to be very useful for measuring protein:carbohydrate interactions. Therefore, we have demonstrated the kinetics and precise affinity K of the ofCS antibody to CS. dTo determine K, we used the Attana biosensor platform (AttanaA200, AttanaAB) using a dual channel system. Briefly, sensor chips were coated with recombinant streptavidin (50 μg / ml) using EDC / S-NHS according to the manufacturer's protocol. Biotinylated ofCSPGs were then flowed over the A channel chip, giving a baseline shift indicative of binding. The B channel chip was left blank as a control. The baseline was stabilized by flowing running buffer (PBS) over the chip. Increasing concentrations of antibody fragments (3.125-100 nM) were flowed over the chip at a flow rate of 20 μl / min. Nonspecific binding was subtracted using a non-coated chip run in parallel. Chips were regenerated between sample runs and data was analyzed using Attester Evaluation (Attana) and / or TraceDrawer (Ridgeview) software. Chip on- and off-rates were used to determine K. d The values ​​were evaluated and plotted in Table 5.

[0207] [Table 6]

[0208] In summary, all phage display-derived antibody fragments tested showed very high affinity for ofCSPGs in the low nanomolar range. Similar high affinity and high specificity for ofCSs were also seen with other antibody formats. This is a surprising finding, since carbohydrate interactions are usually of low affinity and low specificity.

[0209] To test these other formats, monomers, dimers, and tetramers were constructed, either by genetic fusion or by conjugation to a bivalent Spycatcher format via spytag. Full IgG and scFV-FcIgG were also constructed. All formats bound ofCS with very high affinity, between 0.1 nM and 40 nM (Figure 14). Multimeric constructs had a K of approximately 25 nM. DIt is a more effective binder than the corresponding monomer, which is shown to exhibit values ​​of 0.01 and 0.02. The same figure also shows that the on-rate and off-rate can vary depending on the choice of construct format. More precisely, the K of binding to ofCS by the various formats of antibodies and variants of C9 (i.e., antibodies and variants having the light and heavy chain CDRs set out in SEQ ID NOs: 121-127) is D Experiments designed to determine the value gave the following results: C9 scFv monomer:K D =40nM C9 scFv 2 Tandem dimer: K D =3nM C9 scFv 2 SpyCatcher dimer::K D =1.3nM C9 IgG1:K D =16nM C9 ScFv-Fc:K D =5nM C9 scFv 4 SpyCatcher tetramer:K D =0.1nM

[0210] Thus, depending on the specific intended use of the antibodies and antibody variants of the invention, their binding affinity can be engineered by choosing the appropriate format. In the context of in vivo imaging for the treatment or diagnosis of malignancies, targeting of solid tumors with antibodies and antibody variants can benefit from the use of low molecular weight constructs with high on-rates and off-rates (off-rates are less important in solid tumors compared to low abundance targets due to the high target density). On the other hand, for targeting non-solid malignancies (such as leukemia), constructs with low off-rates and high affinity are more appropriate. Furthermore, various antibody and antibody variant formats differ in terms of biological half-life, and while a long biological half-life may be crucial in many therapeutic settings where tumors are targeted, the opposite may be true in diagnostic settings or for certain treatments, including radiotherapy. Finally, when antibodies and antibody variants are used in vitro in various assays, the avidity of the constructs can be fine-tuned to meet the exact needs of the assay. In other words, the invention allows antibody-derived constructs to be tailored to be particularly suitable for a specific task, in terms of affinity / avidity properties and biological half-life. EXAMPLES

[0211] ofCS antibodies and antibody fragments bind to a broad range of cancer cells in vitro We previously showed that the malaria protein rVAR2, which binds to ofCS, also binds to ofCS present in all cancer cell lines but not to normal cancer cells. The aim in generating an ofCS-specific antibody was to generate a reagent with similar or overlapping specificity to rVAR2, i.e., binding to either cancer cells expressing ofCS or secreted ofCS.

[0212] Cells were grown to 70%–80% confluency in appropriate growth medium and harvested with EDTA detachment solution (Cellstripper). Cells were incubated with proteins (300–25 nM) in PBS containing 2% fetal bovine serum (FBS) for 30 min at 4 °C and binding was analyzed on a FACSCalibur (BD Biosciences) after secondary incubation with anti-V5-FITC antibody. For inhibition studies, proteins were co-incubated with 400 μg / mL CSA (Sigma, St. Louis, MO, USA; catalog no. 27042).

[0213] The data, exemplified by the binding of ofCS IgG antibodies to Karpas lymphoma cells tested by flow cytometry, clearly showed that ofCS IgG binds to cancer cells to various degrees (Figure 4) and that the binding was completely or partially inhibited by the addition of soluble chondroitin sulfate. In all cases the binding was higher than with rVAR2, but this could also be due to the use of different secondary antibodies, since rVAR2 was tested with anti-V5 antibodies and the monoclonal antibodies were tested with anti-human IgG.

[0214] The analysis was then extended to include the scFv format, this time tested in a solid breast cancer tumor cell line (4T1). rVAR2 was included as a benchmark control. All antibody fragments showed significant tumor binding (Table 6), often many-fold higher than rVAR2 and in all cases many-fold higher than the antibody control alone, although F3 only showed a two-fold increase compared to the control.

[0215] [Table 7]

[0216] Again, we wished to compare the panel of ofCS antibodies to available and published antibodies CS56, 2H6, and BE-123. For comparison, B3 IgG was included. The CS56 and 2H6 antibodies only showed slightly higher binding than the negative control, while B3 showed over 100-fold higher binding than the control (Table 7). BE-123 required chondroitinase treatment to show binding.

[0217] [Table 8]

[0218] Selected scFvs were then tested on a larger, more diverse panel of tumor cells. Fresh white blood cells from healthy donors were included in the analysis. Flow cytometry values ​​were scored, with values ​​of 0-1 being background and similar to the antibody's negative control, while values ​​of 2 and above are considered positive binding. It was clear that ofCS antibodies bound to a variety of tumor cells but not to normal white blood cells.

[0219] [Table 9] EXAMPLES

[0220] ofCS antibody fragments bind to tumor and placental tissue sections but not to non-cancerous tissues The binding of the antibody fragments to primary cancer tissues taken from human patients was investigated using immunohistochemistry (IHC). The staining protocol was optimized on the VentanaDiscoveryXT platform without epitope retrieval. Paraffin-embedded tissues spotted on glass slides were incubated with V5-tagged scFv for 1 h at room temperature, washed for 8 min, incubated with 1:700 mouse anti-V5 antibody for 30 min, washed for 8 min. Bound anti-V5 was subsequently detected using UltraMap anti-mouse HRP. All antibody fragments stained tumor tissues, exemplified here by binding to bladder cancer tissue. Furthermore, the antibodies bound to ofCS in placental tissues and no binding to non-cancerous tissues, exemplified here by liver tissue (Figure 5). Staining could be blocked by adding 200 μg / μl CSA or by chondroitinase ABC treatment (data not shown). To demonstrate the ability of the antibodies and antibody variants to stain all types of tumor tissues, tissue microarrays containing malignant tissues from pancreas, breast, colon, stomach, lung, lymph node, liver, skin, and esophagus and corresponding normal adjacent tissues were stained and binding was detected using immunofluorescence. To visualize tissue integrity, nuclei of all sections were stained with DAPI. Figure 12 shows binding using the C9scFv antibody fragment, showing strong binding to all malignant tumor tissues and no binding to normal non-malignant tissues. EXAMPLES

[0221] IVIS tumor localization data Anti-ofCSIgG was NIR-labeled via the available cysteine ​​with Alexa750 using maleimide chemistry. This was done using an excess amount of NIR probe (4 × mol) according to the manufacturer's instructions. The conjugated protein was injected IV (4 mg / kg) into the tail vein of healthy and tumor-bearing mice 10 days after the formation of subcutaneous tumors in the right flank. Mice were scanned using an IVIS Spectrum CT scanner (PerkinElmer). Scans were performed at time intervals ranging from 10 min to 48 h. In vivo tumor signal quantification is presented as absolute signal relative to the signal in the flank of healthy control mice. Data analysis was performed using Living Image Software (Caliper Life Sciences). Anti-ofCSIgG was found to be located in the tumor after several hours and was measurable for at least 24 h (Figure 6). EXAMPLES

[0222] Toxin-conjugated ofCS IgG-in vitro cancer cell killing To investigate whether antibodies can be used to deliver cytotoxic payloads to cancer cells, antibody-drug conjugates (ADCs) were prepared following standard procedures by conjugating the vc-MMAE microtubule inhibitor to cysteines on IgG. Briefly, TCEP was added in 3–6 molar excess to reduce IgG disulfide bonds and incubated at 37 °C for 90 min. The reaction was cooled and an 8 molar excess of Mal-vc-MMAE was added and incubated at +4 °C for 60–90 min. The reaction was stopped by adding an 8 molar excess of cysteine ​​and incubating for 15 min. Free Mal-vc-MMAE was removed using a Zebaspinn column equilibrated with PBS and then concentrated on a Vivaspinn column. The ADCs were quality controlled by SDS-page and HPLC-SEC, and the DAR was measured in the range of 2–4. Furthermore, the ADCs were demonstrated to retain tumor cell binding by flow cytometry and ofCS binding in ELISA after conjugation. Cells were removed from the culture vessel using Gibco® Trypsin-EDTA (Invitrogen #25300-054). Detached cells were diluted to 25,000 cells / mL in the respective growth medium (Invitrogen #: 10313-021, A10491-01, 16600-082, 12561-056, 35050-061, 11415-064) + 10% fetal bovine serum (Corning #: 35-015-CV) and dispensed at 2500 cells / well in 100 μl / well. Cells were seeded in black-walled flat-bottom 96-well plates (Costar #3904). Adherent cell lines were incubated overnight at 37°C in a 5% CO2 atmosphere to allow the cells to attach to the surface of the microtiter plate, and suspension cells were seeded immediately before use. Test compounds were directly diluted in the appropriate cell growth medium to 5x the desired final concentration. These compounds were then titrated 1:3 in eight steps. Controls without test compound (growth medium only) were included in each microtiter plate in sextuplicate. 25 μl / well of prepared titrations were added in triplicate to each cell line analyzed. Cells and titrations were incubated at 37°C / 5% CO2 for 5 nights.After incubation, 30 μl of CellTiter-Glo® was added to each assay well to measure cell viability using prepared CellTiter-Glo® (Promega#G7572) reagent. The mixtures were incubated in the dark for at least 20 minutes, after which luminescence was measured using a microplate luminometer (integration time 500 ms). Collected relative luminescence units (RLU) were converted to % cytotoxicity using the RLU values ​​measured from the growth medium only control as follows: % cytotoxicity = 1 - [well RLU / mean RLU of medium only control]. Data (percent cytotoxicity vs. ADC concentration (log10 [nM])) were plotted and analyzed by nonlinear regression using GraphPad Prism software v.5.02 to obtain EC50 estimates.

[0223] All tested antibodies specific for chondroitin sulfate and conjugated with toxins mediated cell killing with low nanomolar IC50 values, identical to previously published results using rVAR2 drug conjugates. This was demonstrated in both mouse cancer cells (4T1) and a panel of human cancer cells including PC3 prostate, Karpas lymphoma, Colo205 colorectal, and fresh patient-derived cell lines (PDX). Table 9 shows representative ADCs tested in PC3 prostate cancer cell lines and 4T1 breast cancer cell lines. As a negative control, an ADC that does not bind to cancer cells was used, and as a positive control, free soluble MMAE was used, which readily diffuses into cells to mediate cell killing. The VAR2CSA drug conjugate (VDC) was used for benchmarking.

[0224] [Table 10]

[0225] The ability of the drug conjugates to mediate cancer cell killing is further tested in a panel of cancer cell lines representing all types of tumor origins (i.e., cell lines of hematopoietic, epithelial, and mesenchymal origin). EXAMPLES

[0226] Toxin-conjugated ofCSIgG-in vivo tumor cell killing Cancer treatments based on targeting ofCS with antibody panels can take the form of antibody-drug conjugates. To demonstrate the efficacy of anti-ofCSADCs, an animal model of murine colon cancer was established by subcutaneously injecting murine CT26 tumor cells (100,000) into the right flank of immune-competent Balb / c mice. To also test efficacy against human tumors, we established an animal model of murine colon cancer by injecting murine CT26 tumor cells (100,000) into the right flank of immune-competent Balb / c mice. To test efficacy against human tumors, we also inject Karpas lymphoma (1.10 6 The minimum tumor size in both models was 70–120 mm. 3 At the time of reaching 100 mg / kg, mice were randomly divided into three groups and received a total of three intravenous injections of vehicle (saline), control ADC, or ofCSADC, each at a dose of 3 mg / kg. Tumor growth was monitored using a caliper measurement tool to measure the three longest perpendicular axes in the x / y / z plane of each tumor. Tumor volume was calculated according to the standard formula: volume = xy2 x 0.5236. Mice were weighed three times a week to monitor acute toxic effects. In the CT26 colon cancer model, tumors completely regressed in all treated animals, whereas in the control group, 6 out of 7 mice reached the endpoint with large tumors at day 20 (see Figure 10). Mice treated with ofCSADC remained tumor-free throughout the entire study (4 months) and were resistant to challenge with new tumors. The same was true for Karpas (see Figure 11), with regression observed in all mice and 4 out of 6 cured. All animals in the treatment groups gained weight during the study and no signs of acute or long-term toxicity were observed.

[0227] Given this background, it is expected that the treatment regimen will be well tolerated in humans and will be effective, with tumor growth either halted completely or regressing in size after treatment. EXAMPLES

[0228] ofCS antibodies inhibit cancer cell migration and tumor dissemination and metastatic spread in vivo We wanted to determine whether targeting ofCS with antibodies could inhibit tumor metastasis in vivo. To this end, we established two animal models to investigate two key events in metastatic spread: cell colonization (or seeding) and tumor implementation. In the 4T1 seeding model, we used 5 × 10 cells suspended in 100 μL of 100 nmol / L ofCS-specific antibodies or saline or isotype-matched control antibodies. 5 Luciferase-labeled cells were injected into the tail vein of immune-competent mice. Animals were monitored up to 7 weeks after injection using an IVIS imaging system. Mice were sacrificed when they reached a predefined humane endpoint. For the B16 melanoma model (tumor mounting model), 5 × 10 cells in 100 μL PBS were 5 B16-F10GP cells were injected into the right flank of C57BL / 6 mice. Animals were randomly divided into two groups of 10 mice. One group was treated with 100 μg of CS antibody injected intravenously on days 0, 6, and 9. A control group was treated with an equal volume of PBS, and the other control group was treated with an isotype-specific control antibody. Tumor size was monitored by measuring the two longest perpendicular axes of the x / y plane of each tumor, measured manually using a caliper measuring tool. Tumor volume was calculated according to the standard formula: volume = xy2 × 0.5236.

[0229] Preincubation of 4T1 breast cancer cells with ofCS antibodies strongly inhibited colonization of distant organs and significantly extended the life span of treated mice (Figure 7). The same was evident in the treatment of subcutaneous B16 melanoma tumors when performed. Combined with data showing that ofCS antibodies bind to circulating tumor cells (see Example 14), treating patients with antibodies that bind and opsonize circulating cancer cells is highly likely to have a clinically significant impact.

[0230] Furthermore, we plan to test the effect of ofCS antibodies on cancer cell migration and invasion in vitro. We do this by growing tumor cells of different origins to 70% confluency. We then starve them of serum for 24 hours in the presence of 450 nmol / L ofCS or control antibodies. We detach the cells with a Cellstripper and count them in triplicate. We then add 100,000 cells to each insert of a Boyden chamber plate (Chemi-Con). We use separate kits for migration and invasion. The invasion kit contains a membrane coated with a basement membrane extract. We add medium with or without chemoattractants to the bottom wells. We then incubate the plates for 18-36 hours at 37 °C. We measure the number of migrating cells with a fluorescent probe and compare it to a standard curve. We expect that ofCS antibodies will interfere with cell migration. This confirms an important biological role for ofCS antibodies as well as the proposed mechanism that rVAR2 inhibits cell migration by inhibiting the focal adhesion pathway. EXAMPLES

[0231] CART cells targeting ofCS inhibit tumor growth in vitro and in vivo Chimeric antigen receptor (CAR) T cells can be engineered to express anti-ofCS antibody fragments or to present the SpyCatcher protein for binding of Spy-tagged scFV fragments. Here, CARs consisting of the SpyCatcher domain, CD28 and / or CD3 zeta signaling domains, and signaling peptides were introduced into human T cells (immune effector cells) such that the CAR portion of the construct was glycosylated and anchored to the cell membrane of the immune effector cells. CAR-expressing T cells (CART cells) were then mixed with Spy-tagged scFVs against ofCS (here the scFVs are C9 sequences) and mixed with human cancer cells such as LNCap (prostate cancer), U2OS (osteosarcoma), and survival was monitored using an IncuCyte instrument. In both cases, CAR-T cells targeted to ofCS were able to effectively kill tumor cells in vitro, especially tumor cells of very different origins. The negative control, non-transduced T cells mixed with ofCS antibody fragments, had no effect on tumor cells. See Figure 13.

[0232] On the basis of the findings reported here, it is hoped that efficacy will be demonstrated in animal efficacy studies. EXAMPLES

[0233] Immunocytokines: Immunotherapy based on ofCSmAb fused to cytokines We hope to generate immunocytokines consisting of ofCSmAbs fused to cytokines that can target tumors and simultaneously stimulate antitumor immune responses. To this end, human and mouse cancer cells (e.g., MG-63, RH-30, K7M2, NB-16) stably expressing a fluorophore for tracking purposes are co-cultured with peripheral blood mononuclear cells (PBMCs) and treated with an ofCS-targeting scFv fragment fused to a mutant of the immunostimulatory cytokine interleukin-2 (ofCSscFv-IL2v). Specifically, we hope to develop a novel monomeric bispecific ofCS-targeting immunocytokine that contains an IL-2v moiety with reduced binding to the IL-2 receptor a (CD25). We will use an IncuCyte instrument to monitor cancer cell survival and PBMC proliferation in real time. The performance of ofCSscFv-IL2v will be tested in conventional culture (2D) and in a spheroid model that closely resembles the 3D organization of tumor tissue. Compared with commercially available recombinant wild-type IL-2, ofCSscFv-IL2v is expected to have a better ability to kill cancer cells of various origins. In another iteration, we will test the ability of ofCSscFv-IL2v to activate PBMCs by evaluating PBMC proliferation and cytokine secretion similar to equimolar concentrations of IL-2v. Furthermore, we would like to compare the cancer cell binding properties of ofCSscFv-IL2v with that of ofCSscFv. In another iteration, we would like to test the systemic antitumor protection by intratumoral administration of ofCSscFv-IL2v tumors in vivo. Furthermore, we will test whether the potential antitumor effect can be enhanced by synergy with immune checkpoint inhibitors (anti-PD-L1 / PD-1 or anti-CTLA4). EXAMPLES

[0234] Immunotherapy based on ofCSmAb fused to anti-CD3 eradicates tumors in vivo This study was conducted to test whether scFv fragments of the ofCS antibody fused to a mouse anti-CD3 antibody could cure cancer in mice with fully functional immune systems. Mouse cell lines 4T1 (breast cancer), B16-F10 (melanoma), CT26 (colorectal cancer), and TC-1 (derived from primary lung epithelial cells) were used for in vitro and in vivo experiments, the latter injected into C57BL / 6J or BALB / cAnNRJ (BALB / c) mice. Mice were either completely randomly assigned to groups or divided into groups based on size, ensuring equal average tumor size before treatment. All experiments used C57BL / 6J and BALB / cAnNRJ (BALB / c) mice purchased from Janvierlabs. Mice were 6–8 weeks old on arrival and housed for at least 1 week before use.

[0235] C57BL / 6J mice were injected subcutaneously with 100,000 B16-F10 cells in 100 μL PBS in the left lower quadrant or right flank of the abdomen. BALB / c mice were injected subcutaneously with 75,000 4T1 cells or 500,000 CT26 cells in 100 μL PBS in the right flank. All mice were treated peritumorally (PT) with 12 μg CSscFV-aCD3Mu in a total volume of 50 μL in PBS. 100 μg mouse anti-mCTLA-4 (Invivofit) was administered intraperitoneally (IP) three times over one week, with the first dose being doubled. 100 μg InvivoPlus anti-mouse PD-1 (BioXCell) was administered IP twice during the first week.

[0236] The ofCSscFV-aCD3Mu molecule maintained binding of each moiety as assessed by flow cytometry and induced effective killing of cancer cells in vitro when preactivated splenocytes were added. In vivo, ofCSscFV-aCD3Mu prevented the formation of mouse breast cancer 4T1 tumors when treatment was initiated peritumoral (PT) on day 1 after tumor cell injection (Figure 8). It was also able to slow the growth of larger solid tumors. When ofCSscFV-aCD3Mu was combined with a checkpoint inhibitor (CPI), ofCSscFV-aCD3Mu treatment eliminated solid tumors and metastases in the majority of treated mice. To investigate whether this highly effective treatment regimen was valid beyond 4T1 breast cancer, we established subcutaneous models of B16-F10 malignant melanoma and CT26 colorectal tumors and treated them with a combination of CPI and V-aCD3Mu. In these models, V-aCD3Mu and CPIs also induced complete tumor regression in the majority of treated mice. In summary, our data suggest that ofCS-targeted immunotherapy is effective against a broad range of tumors. This effect is enhanced by combining with CPIs, which induces an abscopal response that results in complete responses and renders mice resistant to tumor rechallenge. EXAMPLES

[0237] Specific capture of ultra-rare cancer cells in blood samples for diagnostic purposes Context: Circulating tumor cells (CTCs) are malignant cells that detach from solid tumor compartments and enter lymphatic or blood circulation. The presence of CTCs in blood is associated with poor patient prognosis, but offers the opportunity for continuous and non-invasive access to information on tumor evolution and progression. This could not only allow for earlier detection of cancer but also provide the basis for better therapeutic decisions. Unfortunately, CTCs are extremely rare compared to the vast numbers of normal white blood cells. Moreover, CTCs are characterized by a high degree of heterogeneity and cellular plasticity, which complicates their specific and sensitive isolation.

[0238] Methods: Flow cytometry analysis was applied to measure the binding of the different antibody constructs to cancer cells or healthy white blood cells (WBCs). From fresh blood drawn from healthy donors, WBCs were isolated by simple red blood cell lysis followed by a washing step. Colon cancer cells (COLO205) were harvested from cell culture and both cell types were incubated with 150 nM of the respective constructs. The scFv constructs containing SpyTag were dimerized by SpyCatcher dimers containing a HIS tag and binding was detected using an anti-HISFITC antibody. To investigate whether the antibody constructs could successfully bind and isolate a small number of cancer cells spiked in a blood sample, the constructs were dimerized using biotinylated SpyCatcher dimers (in the case of full IgG, the antibodies were directly biotinylated). After carefully mixing 100 pre-stained cancer cells into 3 mL of blood, the blood sample was treated by red blood cell lysis and subsequently incubated with approximately 100 nM of the respective constructs. Finally, they added streptavidin-coated magnetic beads, which allow high-affinity binding between the biotinylated antibody structures and the magnetic beads. Using a simple magnet, they separated the antibody-bound cancer cells from a large number of healthy white blood cells and counted them under a microscope.

[0239] result: As shown in Table 10, the various structures bound at least 100 times more strongly to cancer cells compared to healthy white blood cells. Because a patient's blood sample may contain only a few CTCs in a background of millions of white blood cells, the ability of the antibody to distinguish between target cells (cancer cells) and non-target cells is of great importance.

[0240] The high degree of specificity was further tested by conjugating the F4scFv to a SpyCatcher dimer labeled with a strong fluorophore and using the construct to stain cancer cells after spiking them into blood. When the samples were analyzed under a microscope, COLO205 colon cancer cells were brightly and vividly stained, with little staining of surrounding healthy white blood cells.

[0241] The antibody constructs were also tested for their ability to separate cancer cells from blood samples. To allow the cancer cells to be identified, these cells were pre-stained with a fluorescent dye. 100 cancer cells were spiked into 3 mL of blood and processed as described above. Different types of cancer cells were tested. After antibody-based magnetic separation, microscopic analysis revealed the number of captured cancer cells. The number of captured cells relative to the number of cells spiked into the sample constituted the recovery rate (percentage) and is shown in Table 11. The antibody constructs successfully captured different types of cancer cells from blood.

[0242] [Table 11] [Table 12]

[0243] Conclusion: In conclusion, the wide range of antibody structures tested (scFv, Fab, and full IgG) demonstrated the ability to specifically bind to cancer cells even when these target cells were found within a very high background of normal non-target cells such as white blood cells. This opens the possibility of utilizing antibody structures to capture rare circulating tumor cells (CTCs) from blood samples of cancer patients, thus offering many new diagnostic opportunities. EXAMPLES

[0244] Specific capture of proteoglycans in diagnostic liquid biopsies. We demonstrated that tumors overexpress CS-modified proteoglycans. Mass spectrometry of proteoglycans extracted from urine, plasma, and tumor biopsies using either VAR2 or anti-ofCS antibodies identifies a variety of of-CS-modified proteins, including agrin, biglycan, CD44, decorin, glypican, endorepellin, integrin beta-1, laminin subunit gamma-2, neuropilin-1, syndecan, testoscan, sushi repeat-containing protein, CSPG4, endocan, and versican. Using a combination of VAR2, ofCS antibodies, and / or antibodies specific for the proteoglycan protein core, we developed a method to detect CS-modified proteoglycans in body fluids, including plasma, cerebrospinal fluid, and urine. In this example, anti-ofCS antibodies were immobilized on an ELISA plate. Different concentrations of endocan were then added to the sample buffer, and antibodies specific for the protein core of CSPGs were used to detect the levels of proteoglycans captured by CS (Figure 9). Such an assay has a clinically relevant detection range down to 1 ng of endocan. Optionally, the plasma sample can be enriched with GAGs prior to analysis using the IEX column. EXAMPLES

[0245] Binding groove characteristics CS-binding scFvs share a common feature and are predicted to have a positively charged binding groove defined by alpha-fold structure prediction, which resembles the positive groove crucial for VAR2CSA binding to ofCS (Wang et al., Nat. Commun., 2021). The scFv binding groove is defined by the following criteria and applies to the structural class of antibodies with flexibility in the variable loop sequences:

[0246] a) CDR-L2 and CDR-H3, defined using the KABAT scheme, contain multiple lysine or arginine type amino acid residues. In addition, CDR-L1 and CDR-H2 may also contain lysine or arginine residues. A total of 3–6 residues are present in the analyzed scFv classes, which are surface exposed and form a positive CSA / CSC binding groove. In all cases, the positive residues are V H Domain and V L It is present in both variable domains and forms a groove that intersects both variable domains. b) The positive binding groove is defined by surface-exposed arginine and lysine type amino acid residues in close proximity to each other. This proximity is defined by measuring the distance between the C-α residues. The shortest distance between two adjacent residues in space is 7-10 Å. As an example, for C9scFv, the distance between the C-α:C-α atoms of residues CDR-L3ARG100:CDR-H3ARG229 is 8.4 Å, CDR-H3ARG229:CDR-H3ARG227 is 7.0 Å, CDR-H3ARG229:CDR-L2ARG51 is 10.5 Å, and CDR-L2R51:CDRVLLYS67 is 9.3 Å.

[0247] List of references TIFF2025507321000013.tif194157

Claims

1. An antibody or antibody variant that specifically binds to chondroitin sulfate (CS) glycosaminoglycan chains, and that exhibits higher binding affinity for oncofetal CS (ofCS) than for non-oncofetal glycosaminoglycans such as heparan sulfate and hyaluronic acid.

2. - competes with VAR2CSA (SEQ ID NO: 135) or a CS-binding fragment thereof (such as the polypeptide having SEQ ID NO: 136) for binding to CS glycosaminoglycan chains; and / or - competes with scFv having any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, 121, and 128 for binding to CS glycosaminoglycans; 2. The antibody or antibody variant of claim 1.

3. The binding site is V Ｈ / V Ｌ 2. The antibody or antibody variant of claim 1, characterized by the presence of a positively charged groove parallel to the boundary.

4. The positively charged groove is V Ｌ and V Ｈ 4. The antibody or antibody variant of claim 3, which is constituted by positively charged, surface-exposed amino acid residues present in both variable loops of the domain.

5. The antibody or antibody variant of claim 4, wherein the number of positively charged amino acid residues is 3, 4, 5, or 6.

6. V Ｈ / V Ｌ The antibody or antibody variant of claim 4 or 5, wherein the distance between the alpha carbon atoms of positively charged amino acids that cross the boundary is at most 12 Å, such as in the range of 6-12 Å, preferably 6.7-11.4 Å.

7. The antibody or antibody variant of any one of the preceding claims, which has a higher affinity for CS than the antibodies 2H6, CS56, BE-123 and PG-5.

8. 2. The antibody or antibody variant of any preceding claim which is different from an IgM antibody.

9. An antibody or antibody variant according to any one of the preceding claims, which binds to native ofCS.

10. 2. The antibody or antibody variant of any preceding claim, which is an IgG antibody or is derived from an IgG antibody.

11. 2. The antibody or antibody variant of any one of the preceding claims, which is a human antibody or is derived from a human antibody.

12. and a second antibody that competes with the first antibody for binding to ofCS, the second antibody comprising a heavy chain variable region (V Ｈ ), and having an amino acid sequence present in any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, 121, and 128. Ｌ ) The antibody or antibody variant of any preceding claim.

13. V Ｈ The area is - an amino acid sequence present in or identical to the amino acid stretch linked to the C-terminus of the sequence GGGGGSGGGGSGGGSGGGSGGGGGS (SEQ ID NO: 137) in any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114 and 128, or - an amino acid sequence present in or identical to the amino acid stretch linked to the C-terminus of the sequence GGGGSGGGGSGGGGSGGG (SEQ ID NO: 138) in SEQ ID NO: 121 13. The antibody or antibody variant of claim 12, having the following structure:

14. V Ｌ The area is an amino acid sequence which is present in or is identical to the amino acid stretch bounded at the N-terminus to the sequence GGGGGSGGGGSGGGSGGGSGGGGGS (SEQ ID NO: 137) in any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114 and 128, or - an amino acid sequence present or identical to the amino acid stretch bound at the N-terminus to the sequence GGGGSGGGGSGGGGSGGG (SEQ ID NO: 138) in SEQ ID NO: 121 13. The antibody or antibody variant of claim 12, having the following structure:

15. a light chain complementarity determining region (LCDR) 1 having the sequence of SEQ ID NO: 2, 9, 16, 23, 30, 37, 44, 52, 59, 66, 73, 80, 87, 94, 101, 108, 115, 122, or 129, or a sequence having at least 70% sequence identity to any of these sequences; LCDR2 having the sequence of SEQ ID NO:4, 11, 18, 25, 32, 39, 46, 54, 61, 68, 75, 82, 89, 96, 103, 110, 117, 124, or 131, or a sequence having at least 70% sequence identity with any of these sequences; LCDR3 having the sequence of SEQ ID NO:5, 11, 18, 25, 32, 39, 46, 54, 61, 68, 75, 82, 89, 96, 103, 110, 117, 124, or 131, or a sequence having at least 70% sequence identity with any of these sequences; , 12, 19, 26, 33, 40, 47, 55, 62, 69, 76, 83, 90, 97, 104, 111, 118, 125, or 132, or a sequence having at least 70% sequence identity with any of these sequences. Among these sequences, SEQ ID NOs: 6, 13, 20, 27, 34, 41, 48, 56, 63, 70, 77, 84, 91, 98, 105, 11 HCDR2 having the sequence of SEQ ID NO: 7, 14, 21, 28, 35, 42, 49, 57, 64, 71, 78, 85, 92, 99, 106, 113, 120, 127, or 134, or a sequence having at least 70% sequence identity with any of these sequences.

5. The antibody or antibody variant of any one of the preceding claims, comprising:

16. The following combinations of amino acid sequences for LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3, respectively: SEQ ID NOs: 2, 3, 4, 5, 6, and 7; or SEQ ID NOs: 9, 10, 11, 12, 13, and 14; or SEQ ID NOs: 16, 17, 18, 19, 20, and 21; or SEQ ID NOs: 23, 24, 25, 26, 27, and 28; or SEQ ID NOs: 30, 31, 32, 33, 34, and 35; or SEQ ID NOs: 37, 38, 39, 40, 41, and 42; or SEQ ID NOs: 44, 45, 46, 47, 48, and 49; or SEQ ID NOs: 52, 53, 54, 55, 56, and 57; or SEQ ID NOs: 59, 60, 61, 62, 63, and 64; or SEQ ID NOs: 66, 67, 68, 69, 70, and 71; or SEQ ID NOs: 73, 74, 75, 76, 77, and 78; or SEQ ID NOs: 80, 81, 82, 83, 84, and 85; or SEQ ID NOs: 87, 88, 89, 90, 91, and 92; or SEQ ID NOs: 94, 95, 96, 97, 98, and 99; or SEQ ID NOs: 101, 102, 103, 104, 105, and 106; or SEQ ID NOs: 108, 109, 110, 111, 112, and 113; or SEQ ID NOs: 115, 116, 117, 118, 119, and 120; or SEQ ID NOs: 122, 123, 124, 125, 126, and 127; or SEQ ID NOs: 129, 130, 131, 132, 133, and 134 and a paratope defined by 16. The antibody or antibody variant of claim 15, wherein each sequence can be independently a sequence having at least 70% sequence identity with the indicated SEQ ID NO.

17. 17. The antibody or antibody variant of claim 15 or 16, wherein the sequence identity is selected from at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

18. - a V having an amino acid sequence identical to or present in an amino acid sequence C-terminally linked to the sequence GGGGGSGGGGSGGGSGGGSGGGGGS (SEQ ID NO: 137) of any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, and 128; Ｈ Area, and - a V having an amino acid sequence identical to or present in an amino acid stretch bound at the C-terminus to the sequence GGGGGSGGGGSGGGSGGGSGGGGGS (SEQ ID NO: 137) of any one of SEQ ID NOs: 1, 8, 15, 22, 29, 36, 43, 50, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114 and 128; Ｈ Area, and - V having an amino acid sequence identical to or present in an amino acid stretch bound at the N-terminus to the sequence of the same SEQ ID NO: GGGGSGGGGSGGGSGGGSGGGGGS (SEQ ID NO: 137); Ｌ region, or - V present in or having an amino acid sequence identical to the amino acid stretch bound at its C-terminus to the sequence GGGGSGGGGGSGGGGGSGGG Ｈ SEQ ID NO: 121 of the region (SEQ ID NO: 138), and - V having an amino acid sequence identical to or present in the amino acid sequence linked to the N-terminus of the sequence GGGGSGGGGGSGGGGGSGGG (SEQ ID NO: 138) of the same SEQ ID NO: 121 Ｌ region Including a combination of Here, V Ｈ and V Ｌ 2. The antibody or antibody variant of any one of the preceding claims, wherein each of the sequences may independently have at least 80% sequence similarity to the indicated SEQ ID NO.

19. 19. The antibody or antibody variant of claim 18, wherein the sequence identity is selected from at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

20. 21. The antibody or antibody variant of any one of claims 1 to 20, wherein the CS glycosaminoglycan chain comprises an N-acetylgalactosamine (GalNAc) residue bearing a sulfate group at the C-4 position in >50%, such as >60%, preferably >70% of the disaccharide repeats of the chain.

21. The antibody or antibody variant of claim 20 , wherein the CS chain comprises 4O-sulfated GalNAc and iduronic acid (IdoA).

22. Equilibrium dissociation constant (K Ｄ ) <10 nM, such as <5 nM, ofCS glycosaminoglycan chains.

23. 10. The antibody or antibody variant of claim 1, wherein the ofCS glycosaminoglycan chains are attached to a protein core forming a chondroitin sulfate proteoglycan (CSPG) present in secreted form on the cell membrane or in the extracellular matrix.

24. 24. The antibody or antibody variant of claim 23, wherein the CSPG is selected from any one of brain natriuretic peptide B, endothelial cell specific molecule 1, Sushi repeat containing protein SRPX, decorin, protein AMBP, biglycan, myeloid proteoglycan, syndecan-4, amyloid-like protein 2, HLA class II histocompatibility complex gamma chain, chondroitin sulfate proteoglycan 4, agrin, testoscan 1-3, neuropilin, CD44 antigen, glypican-1-6, syndecan-1-34, laminin subunit gamma 2, carbonic anhydrase 9, aggrecan, perlecan, collagen alpha-1(XII), collagen alpha-1(XV), collagen alpha-1(XVIII), laminin subunit alpha-4, matrix remodeling associated protein 5, nidogen-2, and versican.

25. The antibody mutants are Fab, Fab', Fab-SH, F(ab) ２ , F(ab′) ２ , ScFv, Fv fragment, heavy chain Ig (such as llama or camel Ig), IgY, V ＨＨ 2. The antibody or antibody variant of any one of the preceding claims, selected from a fragment, a dsFV, a minibody, a diabody, a triabody, a kappabody, an IgNAR, a tandAb, a BiTE, and a multispecific antibody.

26. 2. The antibody or antibody variant of claim 1 , wherein the antibody variant is a bispecific antibody.

27. 27. The antibody or antibody variant of claim 26, wherein the bispecific antibody specifically binds to CD3.

28. A conjugate or fusion protein comprising at least a first and a second moiety, the first moiety being an antibody or antibody variant according to any one of the preceding claims, and the second moiety being a molecule or polypeptide; wherein the first moiety is conjugated or genetically fused to the second moiety, and the second moiety provides or improves a therapeutic and / or diagnostic function of the conjugate or fusion protein.

29. 29. The conjugate or fusion protein of claim 28, wherein the second moiety is selected from any one of a toxin or a fragment thereof, an immunomodulatory molecule or a fragment thereof, a nanoparticle, a radionuclide or a radionuclide-containing material, and a label.

30. 30. The conjugate or fusion protein of claim 29, wherein the second moiety is a toxin.

31. 31. The conjugate of claim 30, wherein the toxin is selected from cytotoxic or cytostatic agents such as alkylating agents, antimetabolites, anti-microtubule agents such as monomethylauristatin E (MMAE), topoisomerase inhibitors such as exetecan, and cytotoxic antibiotics.

32. 29. The conjugate or fusion peptide of claim 28, wherein the second portion is a polypeptide and the first portion is genetically fused to the second portion.

33. 33. The conjugate of claim 32, wherein the second moiety is a cytokine or chemokine, such as interleukin-2 (IL-2), interleukin-7 (IL-7), interleukin-12, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), or TNF-related apoptosis-inducing ligand (TRAIL).

34. i. a first polypeptide domain that is the transmembrane domain and endodomain of a chimeric antigen receptor (CAR), and ii. A second polypeptide domain which is an antibody or antibody variant according to any one of claims 1 to 27. A polypeptide comprising:

35. A CAR-T cell comprising the polypeptide of claim 34.

36. An isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antibody variant of any one of claims 1 to 27, the conjugate or fusion protein of claims 28 to 32, or the polypeptide of claim 34.

37. 37. A vector, such as an expression vector or a cloning vector, comprising the isolated nucleic acid molecule of claim 36.

38. 38. A host cell comprising or transformed with the vector of claim 37.

39. A method for producing an antibody or antibody variant according to any one of claims 1 to 27, a conjugate or fusion protein according to any one of claims 29 to 32, or a polypeptide according to claim 34, comprising the steps: - Transfecting or transforming a host cell with the vector of claim 37; - expressing the nucleotide sequence according to claim 36, and - isolating the antibody or antibody variant, conjugate or fusion protein, or polypeptide A method comprising:

40. A pharmaceutical composition comprising the antibody or antibody variant of any one of claims 1 to 27, the conjugate or fusion protein of any one of claims 28 to 32, or the CAR-T cell of claim 35, and a pharma- ceutically acceptable carrier, vehicle or diluent.

41. Use of an antibody or antibody variant according to any one of claims 1 to 27, or a conjugate or fusion protein according to any one of claims 28 to 32, for the in vitro detection and / or isolation of cancer cells, such as cancer cells from a subject, such as circulating tumor cells.

42. 42. The use of claim 41, wherein the cancer cells are from a cancer selected from an epithelial tumor, a non-epithelial tumor, and a mixed tumor.

43. 43. The use according to claim 42, wherein the epithelial tumor is a carcinoma or adenocarcinoma, and the non-epithelial tumor or mixed tumor is a liposarcoma, a fibrosarcoma, a chondrosarcoma, an osteosarcoma, a leiomyosarcoma, a rhabdomyosarcoma, a glioma, a neuroblastoma, a medulloblastoma, a malignant melanoma, a malignant meningioma, a neurofibrosarcoma, a leukemia, a myeloproliferative disorder, a lymphoma (such as a B-cell or T-cell lymphoma), angiosarcoma, Kaposi's sarcoma, a malignant teratoma, a dysgerminoma, a seminoma, or a choriosarcoma melanoma.

44. 44. The use according to claim 42 or 43, wherein the cancer is a cancer of the eye, nose, mouth, tongue, pharynx, esophagus, stomach, colon, rectum, bladder, ureter, urethra, kidney, liver, pancreas, thyroid, adrenal gland, breast, skin, central nervous system, peripheral nervous system, meninges, vasculature, testes, ovaries, uterus, cervix, spleen, bone, or cartilage.

45. Use of an antibody or antibody variant according to any one of claims 1 to 27, a conjugate or fusion protein according to any one of claims 28 to 32, a CAR-T cell according to claim 35, or a pharmaceutical composition according to claim 40 for use as a medicament.

46. An antibody or antibody variant according to any one of claims 1 to 27, or a conjugate or fusion protein according to any one of claims 28 to 32, or a pharmaceutical composition according to claim 40, for use in the diagnosis of cancer in a subject.

47. The antibody or antibody variant of any one of claims 1 to 27, the conjugate or fusion protein of any one of claims 28 to 32, the CAR-T cell of claim 35, or the pharmaceutical composition of claim 40, for use in the treatment of cancer in a subject in need thereof, or for use in the treatment of a condition associated with expression (such as inappropriate expression) of ofCS, such as a condition selected from the group consisting of arthritis, arthropathy, multiple sclerosis, healing after nerve injury, cartilage repair, wound healing, and psoriasis in a subject in need thereof, or for use in delivering a therapeutic agent to the placenta in a subject in need thereof.

48. 48. The antibody or antibody variant for use according to claim 46 or 47, wherein the subject is a mammal, such as a dog, cat, horse or human.

49. The antibody or antibody variant, conjugate or fusion protein, CAR-T cell or pharmaceutical composition for use according to any one of claims 46 to 48, wherein cancer is as defined in any one of claims 42 to 44.

50. 23. A method of treating cancer in a subject in need thereof, or a method of treating a condition associated with expression (e.g. inappropriate expression) of ofCS, such as a condition selected from the group consisting of arthritis, arthropathy, multiple sclerosis, healing after nerve injury, cartilage repair, wound healing, and psoriasis, or a method for use in delivering a therapeutic agent to the placenta, comprising administering to the subject a therapeutically effective amount of the antibody or antibody variant of any one of claims 1 to 27, the conjugate or fusion protein of any one of claims 28 to 32, the CAR-T cell of claim 35, or the pharmaceutical composition of claim 40.

51. The method of claim 50, wherein the cancer is a cancer as defined in any one of claims 42 to 44 and / or the subject has a cancer as defined in claim 48.

52. 1. A method for detecting ofCS in a sample, the method comprising: 1) contacting said sample with an antibody or antibody variant according to any one of claims 1 to 27, allowing the antibody or antibody variant to specifically bind to ofCS, and subsequently contacting either the antibody or antibody variant and the ofCS bound thereby with an agent capable of binding to ofCS, allowing said agent to bind to ofCS; or 2) contacting said sample with an agent capable of binding to ofCS, allowing the agent to bind to ofCS, and subsequently contacting either the agent and the ofCS bound thereby with an antibody or antibody variant according to any one of claims 1 to 27, allowing the antibody to bind to ofCS; and quantitatively or qualitatively detecting the presence of a complex comprising the ofCS, an antibody or antibody variant, and a drug. wherein the agent and the antibody or antibody variant are non-competitive for binding to the CS.

53. 53. The method of claim 52, wherein the sample is a sample comprising or consisting of cells and / or tissues, preferably body fluids such as blood, urine, saliva, CS fluid, and lymph; faeces; and biopsies.

54. 53. The method of option 1 of claim 52, wherein the agent is an antibody or antibody fragment according to any one of claims 1 to 27, a protein comprising or consisting of VAR2CSA (SEQ ID NO: 135) or a CSA-binding fragment thereof, e.g., SEQ ID NO: 136, an antibody that binds to chondroitin sulfate, an antibody that binds to the protein core of a proteoglycan, or a glycosaminoglycan staining dye.

55. 53. The method of option 2 of claim 52, wherein the agent is VAR2CSA (SEQ ID NO: 1) or a CSA-binding fragment thereof, or an antibody that binds to the protein core of a proteoglycan.

56. 55. The method of claim 54, wherein the agent is an antibody capable of binding to the protein core of a proteoglycan.

57. The method of any one of claims 52 to 56, wherein the antibody or the drug is bound to a solid or semi-solid phase.

58. 58. The method of any one of claims 52 to 57, wherein detection of the complex is facilitated by labelling the antibody or agent with a detectable moiety or by adding a detectable agent which binds to the antibody or agent.

59. A detection kit comprising an antibody according to any one of claims 1 to 27, an agent as defined in claim 52 or 54, and a reaction vessel, and optionally also a labelling agent which binds to the antibody or the agent.

60. A method of providing a nucleic acid encoding an antibody or antibody variant according to any one of claims 1 to 27, comprising screening a library comprising antigen-binding fragments of the antibody, wherein the library is comprised of display agents selected from cells, viruses and phages comprising nucleic acid fragments encoding the antibody, for binding to a capture agent of the display agent, wherein the capture agent consists essentially of purified ofCS or ofCS-decorated protein, and then isolating the nucleic acid fragments encoding the antibody from the capture agent-bound display agent, and optionally sequencing the isolated nucleic acid fragments.

61. 61. The method of claim 60, wherein the display agent is a yeast cell or a phage selected from phages M3, fd, T4, T7 and lambda.

62. Antigen-binding fragments of antibodies include scFv, F(ab), F(ab') ２ and F(ab').

63. The method of any one of claims 60 to 62, wherein the antigen-binding fragment comprises a synthetic CDR, a semi-synthetic CDR, or a CDR derived from a human antibody library.

64. A method for producing the antibody or antibody variant of any one of claims 1 to 27, comprising expressing in a host cell in culture an expression vector comprising the nucleic acid fragment obtained according to the method of claim 60, and then isolating the expression product from the culture.

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